Disease resistant brassica plants

ABSTRACT

The present invention relates to a mutant  Brassica  plant, which is resistant to a pathogen of viral, bacterial, fungal or oomycete origin. The mutant  Brassica  plant has a reduced level, reduced activity or complete absence of DMR6-1 protein and DMR6-2 protein as compared to a wild type  Brassica  plant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 15/975,670, filed May 9, 2018, which is acontinuation application of U.S. patent application Ser. No. 15/190,675filed Jun. 23, 2016 and issued as U.S. Pat. No. 9,994,861, which is adivisional application of U.S. patent application Ser. No. 14/528,707,filed Oct. 30, 2014 and issued as U.S. Pat. No. 9,546,373, which is adivisional application of U.S. patent application Ser. No. 14/250,875,filed Apr. 11, 2014 and issued as U.S. Pat. No. 9,121,029, which is adivisional application of U.S. patent application Ser. No. 12/525,236,internationally filed Jan. 30, 2008 and issued as U.S. Pat. No.8,742,207, which is a U.S. National Phase application under 35 U.S.C. §371 of International Application No. PCT/EP2008/000718, filed Jan. 30,2008, which claims priority to International Application No.PCT/EP2007/050976, filed Feb. 1, 2007, each of which is incorporatedherein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 701802011721SEQLIST.TXT,date recorded: Jan. 17, 2019, size: 136 KB).

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to disease resistant plants, in particularplants resistant to organisms of the kingdom Fungi and the phylumOomycota, the oomycetes. The invention further relates to plant genesconferring disease resistance and methods of obtaining such diseaseresistant plants for providing protection to Oomycota pathogens.

Description of Related Art

Resistance of plants to fungal and oomycete pathogens has beenextensively studied, for both pathogen specific and broad resistance. Inmany cases resistance is specified by dominant genes for resistance.Many of these race-specific or gene-for-gene resistance genes have beenidentified that mediate pathogen recognition by directly or indirectlyinteracting with avirulence gene products or other molecules from thepathogen. This recognition leads to the activation of a wide range ofplant defense responses that arrest pathogen growth.

In plant breeding there is a constant struggle to identify new sourcesof mostly monogenic dominant resistance genes. In cultivars with newlyintroduced single resistance genes, protection from disease is oftenrapidly broken, because pathogens evolve and adapt at a high frequencyand regain the ability to successfully infect the host plant.

Therefore, the availability of new sources of disease resistance ishighly needed.

Alternative resistance mechanisms act for example through the modulationof the defense response in plants, such as the resistance mediated bythe recessive mlo gene in barley to the powdery mildew pathogen Blumeriagraminis f. sp. hordei. Plants carrying mutated alleles of the wildtypeMLO gene exhibit almost complete resistance coinciding with the abortionof attempted fungal penetration of the cell wall of single attackedepidermal cells. The wild type MLO gene thus acts as a negativeregulator of the pathogen response. This is described in WO9804586.

Other examples are the recessive powdery mildew resistance genes, foundin a screen for loss of susceptibility to Erysiphe cichoracearum. Threegenes have been cloned so far, named PMR6, which encodes apectate-lyase-like protein, PMR4 which encodes a callose synthase, andPMR5 which encodes a protein of unknown function. Both mlo and pmr genesappear to specifically confer resistance to powdery mildew and not tooomycetes such as downy mildews.

Broad pathogen resistance, or systemic forms of resistance such as SAR,has been obtained by two main ways. The first is by mutation of negativeregulators of plant defense and cell death, such as in the cpr, lsd, andacd mutants of Arabidopsis. The second is by transgenic overexpressionof inducers or regulators of plant defence, such as in NPR1overexpressing plants.

The disadvantage of these known resistance mechanisms is that, besidespathogen resistance, these plants often show detectable additional andundesirable phenotypes, such as stunted growth or the spontaneousformation of cell death.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a form of resistancethat is broad, durable and not associated with undesirable phenotypes.

In the research that led to the present invention, an Arabidopsisthaliana mutant screen was performed for reduced susceptibility to thedowny mildew pathogen Hyaloperonospora parasitica. EMS-mutants weregenerated in the highly susceptible Arabidopsis line Ler eds 1-2. Eightdowny mildew resistant (dmr) mutants were analyzed in detail,corresponding to 6 different loci. Microscopic analysis showed that inall mutants H. parasitica growth was severely reduced. Resistance ofdmr3, dmr4 and dmr5 was associated with constitutive activation of plantdefense. Furthermore, the dmr3 and dmr4, but not dmr5 mutants, were alsoresistant to Pseudomonas syringae and Golovinomyces orontii.

In contrast, enhanced activation of plant defense was not observed inthe dmr1, dmr2, and dmr6 mutants. The results of this research have beendescribed in Van Damme et al. (2005) Molecular Plant-MicrobeInteractions 18(6) 583-592. This article does not disclose theidentification and characterization of the DMR genes.

The dmr6 mutant was identified in a loss-of-susceptibility screen in theArabidopsis Ler eds 1-2 background. The DMR6 gene now has been clonedand characterized. Thus, it was found that DMR6 is the gene At5g24530,encoding for an oxidoreductase (DNA and amino acid sequence are depictedin FIG. 2). Oxidoreductases are enzymes that catalyze the transfer ofelectrons from one molecule, the oxidant, to another, the reductant.According to the present invention, it has been found that lack of afunctional DMR6 protein results in downy mildew resistance.

The present disclosure thus provides a plant, such as a Brassica plantwhich is resistant to a pathogen of viral, bacterial, fungal or oomyceteorigin, characterized in that the plant has a reduced level, reducedactivity or complete absence of the DMR6 protein as compared to a plantthat is not resistant to the said pathogen. In some embodiments, theBrassica plant is a plant of the family Brassicaceae (i.e., Cruciferae).In some embodiments, the plant is Brassica oleracea. In someembodiments, the oomycete pathogen is Hyaloperonosporaparasitica/Hyaloperonospora brassicae. In some embodiments, thebacterial pathogen is Xanthomonas campestris campestris.

This form of resistance is in particular effective against pathogens ofthe phylum Oomycota, such as Albugo, Aphanomyces, Basidiophora, Bremia,Hyaloperonospora, Pachymetra, Paraperonospora, Perofascia,Peronophythora, Peronospora, Peronosclerospora, Phytium, Phytophthora,Plasmopara, Protobremia, Pseudoperonospora, Sclerospora, Viennotiaspecies, as well as to pathogens belonging to the Fungi. In someembodiments, the pathogen of the phylum Hyaloperonospora isHyaloperonospora parasitica/Hyaloperonospora brassicae.

The resistance according to the invention is based on an altered, inparticular a reduced level, reduced activity or complete absence of theDMR6 protein in planta. The term “DMR6 protein” in this respect relatesto the DMR6 gene product, such as the protein encoded by the At5g24530gene in Arabidopsis. Such alterations can be achieved in various ways.

In one embodiment of the invention, the reduced level of DMR6 protein isthe result of a reduced endogenous DMR6 gene expression. Reducing theexpression of the DMR6 gene can be achieved, either directly, e.g., bytargeting DMR6, or indirectly by modifying the regulatory sequencesthereof, or by stimulating repression of the gene. In some embodiments,endogenous DMR6 gene expression may be reduced by any suitablemethodology including, without limitation, gene silencing, RNAinterference (RNAi), virus-induced gene silencing (VIGS), smallRNA-mediated post-transcriptional gene silencing, transcriptionactivator-like effector nuclease (TALEN) gene editing techniques,clustered Regularly Interspaced Short Palindromic Repeat (CRISPR/Cas9)gene editing techniques, and zinc-finger nuclease (ZFN) gene editingtechniques.

Modulating the DMR6 gene to lower its activity or expression can beachieved at various levels. First, the endogenous gene can be directlymutated. This can be achieved by means of a mutagenic treatment.Alternatively, a modified DMR6 gene can be brought into the plant bymeans of transgenic techniques or by introgression, or the expression ofDMR6 can be reduced at the regulatory level, for example by modifyingthe regulatory sequences or by modulating gene expression by, forexample, gene silencing, RNA interference (RNAi), virus-induced genesilencing (VIGS), small RNA-mediated post-transcriptional genesilencing, transcription activator-like effector nuclease (TALEN) geneediting techniques, clustered Regularly Interspaced Short PalindromicRepeat (CRISPR/Cas9) gene editing techniques, or zinc-finger nuclease(ZFN) gene editing techniques.

In another embodiment of the present disclosure, the reduced level ofDMR6 protein is the result of a mutation in the DMR6 gene resulting in areduced DMR6 expression as compared to the wild-type DMR6 gene whereinno such mutation is present, or resulting in a reduced mRNA or proteinstability. In a particular embodiment this is achieved by mutations inthe DMR6 coding sequence that result in a non-functional DMR6 protein,i.e. without or with reduced enzymatic activity.

In another embodiment of the invention, reduced expression can beachieved by down-regulation of DMR6 gene expression either at thetranscriptional or the translational level, e.g. by gene silencing or bymutations that affect the expression of the DMR6 gene.

This invention is based on research performed on resistance toHyaloperonospora parasitica in Arabidopsis but is a general concept thatcan be more generally applied in plants, in particular in crop plantsthat are susceptible to infections with pathogens, such as Oomycota andFungi.

The invention is suitable for a large number of plant diseases caused byoomycetes such as, but not limited to, Bremia lactucae on lettuce,Peronospora farinosa on spinach, Pseudoperonospora cubensis on membersof the Cucurbitaceae family, e.g. cucumber and melon, Peronosporadestructor on onion, Hyaloperonospora parasitica on members of theBrassicaceae family, e.g. cabbage, Plasmopara viticola on grape,Phytophthora infestans on tomato and potato, and Phytophthora sojae onsoybean.

When the modification of DMR6 gene expression in a plant is to beachieved via genetic modification of the DMR6 gene or via theidentification of mutations in the DMR6 gene, and the gene is not yetknown it must first be identified. To generate pathogen-resistantplants, in particular crop plants, via genetic modification of the DMR6gene or via the identification of mutations in the DMR6 gene, theorthologous DMR6 genes must be isolated from these plant species.

Various methods are available for the identification of orthologoussequences in other plants.

A method for the identification of DMR6 orthologous sequences in a plantspecies, may for example comprise identification of DMR6 ESTs of theplant species in a database; designing primers for amplification of thecomplete DMR6 transcript or cDNA; performing amplification experimentswith the primers to obtain the corresponding complete transcript orcDNA; and determining the nucleotide sequence of the transcript or cDNA.Suitable methods for amplifying the complete transcript or cDNA insituations where only part of the coding sequence is known are theadvanced PCR techniques 5′RACE, 3′RACE, TAIL-PCR, RLM-RACE andvectorette PCR.

Alternatively, if no nucleotide sequences are available for the plantspecies of interest, primers are designed on the DMR6 gene of a plantspecies closely related to the plant of interest, based on conserveddomains as determined by multiple nucleotide sequence alignment, andused to PCR amplify the orthologous sequence. Such primers are suitablydegenerate primers.

Another reliable method to assess a given sequence as being a DMR6ortholog is by identification of the reciprocal best hit. A candidateorthologous DMR6 sequence of a given plant species is identified as thebest hit from DNA databases when searching with the Arabidopsis DMR6protein or DNA sequence, or that of another plant species, using a Blastprogram. The obtained candidate orthologous nucleotide sequence of thegiven plant species is used to search for homology to all Arabidopsisproteins present in the DNA databases (e.g. at NCBI or TAIR) using theBlastX search method. If the best hit and score is to the ArabidopsisDMR6 protein, the given DNA sequence can be described as being anortholog, or orthologous sequence.

DMR6 is encoded by a single gene in Arabidopsis as deduced from thecomplete genome sequence that is publicly available. In the genome ofrice 3 orthologs, and in poplar 2 orthologs have been identified. Inmost other plant species tested so far, DMR6 appears to be encoded by asingle gene, as determined by the analysis of mRNA sequences and ESTdata from public DNA databases. The orthologous genes and proteins areidentified in these plants by nucleotide and amino acid comparisons withthe information that is present in public databases.

Alternatively, if no DNA sequences are available for the desired plantspecies, orthologous sequences are isolated by heterologoushybridization using DNA probes of the DMR6 gene of Arabidopsis oranother plant or by PCR methods, making use of conserved domains in theDMR6 coding sequence to define the primers. For many crop species,partial DMR6 mRNA sequences are available that can be used to designprimers to subsequently PCR amplify the complete mRNA or genomicsequences for DNA sequence analysis.

In a specific embodiment the ortholog is a gene of which the encodedprotein shows at least 50% identity with the Arabidopsis DMR6 protein(At5g24530) or that of other plant DMR6 proteins. In a more specificembodiment the identity is at least 55%, more specifically 60%, evenmore specifically 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%.

Accordingly, certain aspects of the present disclosure relate to amutant Brassica plant, wherein the plant has a reduced activity or areduced level of a DMR6-1 polypeptide as compared to a correspondingwild type Brassica plant, and wherein the mutant Brassica plant has atleast one non-natural mutation introduced into the DMR6-1 gene; andwherein the mutant Brassica plant has a reduced activity or a reducedlevel of a DMR6-2 polypeptide as compared to a corresponding wild typeBrassica plant, and wherein the mutant Brassica plant has at least onenon-natural mutation introduced into the DMR6-2 gene. In someembodiments, the plant exhibits resistance selected from the group ofresistance to Hyaloperonospora parasitica/Hyaloperonospora brassicae,intermediate resistance to Xanthomonas campestris campestris, or anycombination thereof. In some embodiments, the DMR6-1 polypeptide isselected from the group consisting of a polypeptide with at least 85%sequence identity, at least 88% sequence identity, at least 90% sequenceidentity, at least 95% sequence identity, and at least 99% sequenceidentity to SEQ ID NO: 112; and wherein the DMR6-2 polypeptide is apolypeptide selected from the group consisting of a protein with atleast 85% sequence identity, at least 88% sequence identity, at least90% sequence identity, at least 95% sequence identity, and at least 99%sequence identity to SEQ ID NO: 113. In some embodiments, the DMR6-1gene is selected from the group of a nucleotide with at least 85%sequence identity, at least 88% sequence identity, at least 90% sequenceidentity, at least 95% sequence identity, or at least 99% sequenceidentity to SEQ ID NO: 114; and wherein the DMR6-2 gene is selected fromthe group of a nucleotide with at least 85% sequence identity, at least88% sequence identity, at least 90% sequence identity, or least 95%sequence identity, and at least 99% sequence identity to SEQ ID NO: 115.In some embodiments, the non-natural mutation in DMR6-1 is a prematurestop codon, and wherein the non-natural mutation in DMR6-2 is apremature stop codon. In some embodiments, the at least one non-naturalmutation introduced into the DMR6-1 gene reduces an activity or a levelof a DMR6-1 polypeptide as compared to a corresponding wild typeBrassica plant; and wherein the at least one non-natural mutationintroduced into the DMR6-2 gene reduces an activity or a level of aDMR6-2 polypeptide as compared to a corresponding wild type Brassicaplant. In some embodiments, the DMR6-1 polypeptide is selected from thegroup of a polypeptide with at least 85% sequence identity, at least 88%sequence identity, at least 90% sequence identity, at least 95% sequenceidentity, or at least 99% sequence identity to SEQ ID NO: 112; andwherein the DMR6-2 polypeptide is a polypeptide selected from the groupof a protein with at least 85% sequence identity, at least 88% sequenceidentity, at least 90% sequence identity, at least 95% sequenceidentity, or at least 99% sequence identity to SEQ ID NO: 113. In someembodiments, the DMR6-1 gene is selected from the group of a nucleotidewith at least 85% sequence identity, at least 88% sequence identity, atleast 90% sequence identity, at least 95% sequence identity, or at least99% sequence identity to SEQ ID NO: 114; and wherein the DMR6-2 gene isselected from the group consisting of a nucleotide with at least 85%sequence identity, at least 88% sequence identity, at least 90% sequenceidentity, at least 95% sequence identity, or at least 99% sequenceidentity to SEQ ID NO: 115. In some embodiments, the at least onenon-natural mutation that reduces the activity or level of the DMR6-1protein is selected from the group of a premature stop codon introducedinto the DMR6-1 coding sequence, a frameshift mutation introduced intothe DMR6-1 coding sequence, an insertion introduced into the DMR6-1coding sequence, a deletion of a part or a whole of the DMR6-1 codingsequence, an altered amino acid in a conserved domain of the DMR6-1protein, a modified upstream sequence, a mutated promoter element, or anactivated repressor element; and wherein the at least one non-naturalmutation that reduces the activity or level of the DMR6-2 protein isselected from the group of a premature stop codon introduced into theDMR6-2 coding sequence, a frameshift mutation introduced into the DMR6-2coding sequence, an insertion introduced into the DMR6-2 codingsequence, a deletion of a part or a whole of the DMR6-2 coding sequence,an altered amino acid in a conserved domain of the DMR6-2 protein, amodified upstream sequence, a mutated promoter element, or an activatedrepressor element. In some embodiments, the plant is a Brassica oleraceacultivar selected from the group of cabbage, kohlrabi, kale, Brusselssprouts, collard greens, broccoli, Romanesco broccoli, or cauliflower.In some embodiments, the non-natural mutation introduced into the DMR6-1gene is selected from the group of a C to T mutation at a positioncorresponding to nucleotide 181 of reference sequence SEQ ID NO: 114, aC to T mutation at a position corresponding to nucleotide 691 ofreference sequence SEQ ID NO: 114, a G to A mutation at a positioncorresponding to nucleotide 714 of reference sequence SEQ ID NO: 114, ora deletion of five nucleotides starting at a position corresponding tonucleotide 601 of reference sequence SEQ ID NO: 114; and wherein thenon-natural mutation introduced into the DMR6-2 gene is selected fromthe group of a G to A mutation at a position corresponding to nucleotide368 of reference sequence SEQ ID NO: 115, or a deletion of thenucleotide at a position corresponding to nucleotide 600 of referencesequence SEQ ID NO: 115.

In some embodiments, the present disclosure relates to a seed, tissue,or plant part of the Brassica plant of the above embodiments thatincludes a reduced activity or a reduced level of a DMR6-1 polypeptideas compared to a corresponding wild type Brassica plant and a reducedactivity or a reduced level of a DMR6-2 polypeptide as compared to acorresponding wild type Brassica plant, and wherein the seed, tissue, orplant part comprises at least one non-natural mutation in the DMR6-1gene and at least one non-natural mutation in the DMR6-2 gene.

Further aspects of the present disclosure relate to a method forobtaining a mutant Brassica plant including: introducing at least onenon-natural mutation into the DMR6-1 nucleotide coding sequence andintroducing at least one non-natural mutation into the DMR6-2 nucleotidecoding sequence to produce a mutant Brassica plant with a reducedactivity or a reduced level of a DMR6-1 polypeptide as compared to acorresponding wild type Brassica plant and a reduced activity or areduced level of a DMR6-2 polypeptide as compared to a correspondingwild type Brassica plant. In some embodiments, the mutant Brassica plantexhibits resistance selected from the group of resistance toHyaloperonospora parasitica/Hyaloperonospora brassicae, intermediateresistance to Xanthomonas campestris campestris, or any combinationthereof. In some embodiments, the non-natural mutation is achieved by amutagenic treatment, a radiation treatment, or a gene editing technique.In some embodiments, the mutant Brassica plant produced from the abovemethods includes at least at least one non-natural mutation in theDMR6-1 gene and at least one non-natural mutation in the DMR6-2 gene,wherein the plant further comprises a reduced activity or a reducedlevel of a DMR6-1 polypeptide as compared to a corresponding wild typeBrassica plant and a reduced activity or a reduced level of a DMR6-2polypeptide as compared to a corresponding wild type Brassica plant, andwherein the plant exhibits resistance selected from the group ofresistance to Hyaloperonospora parasitica/Hyaloperonospora brassicae,intermediate resistance to Xanthomonas campestris campestris, or anycombination thereof. In some embodiments, the mutant Brassica plantproduced from the above methods includes at least one non-naturalmutation in the DMR6-1 nucleotide coding sequence that reduces anactivity or a level of a DMR6-1 polypeptide as compared to acorresponding wild type Brassica plant; wherein the plant comprises atleast one non-natural mutation in the DMR6-2 nucleotide coding sequencethat reduces an activity or a level of a DMR6-2 polypeptide as comparedto a corresponding wild type Brassica plant; and wherein the plantexhibits resistance selected from the group of resistance toHyaloperonospora parasitica/Hyaloperonospora brassicae, intermediateresistance to Xanthomonas campestris campestris, or any combinationthereof.

In some embodiments, the DMR6-1 polypeptide is selected from the groupof a polypeptide with at least 85% sequence identity, at least 88%sequence identity, at least 90% sequence identity, at least 95% sequenceidentity, or least 99% sequence identity to SEQ ID NO: 112; and whereinthe DMR6-2 polypeptide is a polypeptide selected from the group of aprotein with at least 85% sequence identity, at least 88% sequenceidentity, at least 90% sequence identity, at least 95% sequenceidentity, or at least 99% sequence identity to SEQ ID NO: 113. In someembodiments, the DMR6-1 gene is selected from the group of a nucleotidewith at least 85% sequence identity, at least 88% sequence identity, atleast 90% sequence identity, at least 95% sequence identity, or at least99% sequence identity to SEQ ID NO: 114; and wherein the DMR6-2 gene isselected from the group of a nucleotide with at least 85% sequenceidentity, at least 88% sequence identity, at least 90% sequenceidentity, at least 95% sequence identity, or at least 99% sequenceidentity to SEQ ID NO: 115. In some embodiments, the present disclosurerelates to a seed, tissue, or plant part of the mutant Brassica plantproduced from the above methods.

In some embodiments of any of the above embodiments, the presentdisclosure relates to a plant part, wherein the plant part is a leaf, astem, a root, a flower, a seed, a fruit, a cell, or a portion thereof.In some embodiments, the plant part is a leaf.

In some aspects, the present disclosure relates to a pollen grain or anovule of the plant of any of the above embodiments.

In some aspects, the present disclosure relates to a protoplast producedfrom the plant of any of the above embodiments.

In some aspects, the present disclosure relates to a tissue cultureproduced from protoplasts or cells from the plant of any of the aboveembodiments, wherein the cells or protoplasts are produced from a plantpart selected from the group=of leaf, anther, pistil, stem, petiole,root, root primordia, root tip, fruit, seed, flower, cotyledon,hypocotyl, embryo, or meristematic cell.

In some aspects, the present disclosure relates to a plant seed producedfrom the plant of any of the above embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the office upon request and paymentof the necessary fee.

FIGS. 1A-1D shows the alignment of the amino acid sequences of the DMR6protein of Arabidopsis thaliana (SEQ ID NO: 62) and orthologs fromAquilegia species (SEQ ID NO: 63), Citrus sinensis (SEQ ID NO: 64),Coffea canephora (SEQ ID NO: 65), Cucumis sativus (SEQ ID NO: 67),Gossypium hirsutum (SEQ ID NO: 68), Lactuca sativa (SEQ ID NO: 70),Medicago truncatula (SEQ ID NO: 71), Oryza sativa (SEQ ID NOs. 72-74),Populus trichocarpa (SEQ ID NOs. 75 and 76), Solanum lycopersicum (SEQID NOs. 77 and 78), Sorghum bicolor (SEQ ID NO: 79), Spinacia oleracea(SEQ ID NO: 81), Vitis vinifera (SEQ ID NO: 82), Zea mays (SEQ ID NO:83), and Zingiber officinale (SEQ ID NO: 84), using the CLUSTAL W (1.83)multiple sequence alignment program (EBI). Below the sequences theconserved amino acids are indicated by the dots, and the identical aminoacids are indicated by the asterisk.

FIG. 2 shows the nucleotide (SEQ ID NO: 61) and amino acid sequence (SEQID NO: 62) of the DMR6 gene (At5g24530, gi 42568064, Genbank NM_122361)and protein (gi 15238567, Genbank NP_197841) of Arabidopsis thaliana,respectively.

FIG. 3 shows the nucleotide (SEQ ID NO: 69) and derived amino acidsequence (SEQ ID NO: 70) of the DMR6 ortholog of Lactuca sativa,respectively.

FIG. 4 shows the nucleotide (SEQ ID NO: 80) and derived amino acidsequence (SEQ ID NO: 81) of the DMR6 ortholog of Spinacia oleracea,respectively.

FIG. 5 shows the nucleotide (SEQ ID NO: 66) and derived amino acidsequence (SEQ ID NO: 67) of the DMR6 ortholog of Cucumis sativus andCucumis melo.

FIGS. 6A and 6B show the downy mildew resistance of the Arabidopsis dmr6mutants. FIG. 6A shows quantification of sporangiophores of H.parasitica isolate Waco9, 7 days post inoculation, on the dmr6-1 mutant(BC2, line E37) compared to its parental line Ler eds 1-2 and on thedmr6-2 mutant (FLAG 445D09 T-DNA line) compared to its parental lineWs-4. FIG. 6B shows restoration of susceptibility by complementationwith the At5g24530 gene in the dmr6-1 mutant. H. parasitica spores permg seedling weight were quantified on Ler eds 1-2, dmr6-1 and 5complementation lines (#121, 122, 211,231, and 241).

FIG. 7 shows the structure of the Arabidopsis DMR6 gene and dmr6-1 anddmr6-2 mutations. The DMR6 gene contains four exons and a codingsequence of 1026 bases. The two alleles are indicated; dmr6-1 with abase change in exon 2, and dmr6-2 with a T-DNA insertion into intron 2.

FIG. 8 shows the relative transcript levels of DMR6 in Ler plants eithermock treated or inoculated with a compatible or incompatible H.parasitica isolate. Transcript levels were determined at different dayspost inoculation. The difference in cycle threshold (ACT) values reflectthe number of additional PCR amplification cycles required to reach anarbitrary threshold product concentration as compared to ACTIN2. A lowerACT value indicates a higher transcript level.

FIGS. 9A-9E shows the expression of the DMR6 promoter-reporter(pDMR6::GUS) construct in transgenic Arabidopsis lines, visualized withonly X-gluc as substrate (FIGS. 9D and 9E) or Magenta-X-gluc (FIGS.9A-9C) and trypan blue staining of H. parasitica growth. FIG. 9A showsLer eds 1-2 (pDMR6::GUS) 3 dpi with H. parasitica, Cala2 isolate. FIG.9B shows Col-0 (pDMR6::GUS) 3 dpi with H. parasitica, Waco9 isolate.FIG. 9C shows Ler eds 1-2 (pDMR6::GUS) 3 dpi with H. parasitica, Emoy2isolate. FIG. 9D shows Col-0 (pDMR6::GUS) 3 dp wounding. FIG. 9E showsCol-0 (pDMR6::GUS) 3 dp BTH application.

FIGS. 10A and 10B show the Q-PCR analysis of the transcript levels ofthe genes; At4g14365, At1g14880, ACD6, PR-1, PR-2 and PR-5, selected asup regulated in the dmr6-1 micro array analysis. FIG. 10A showstranscription levels of the six genes in dmr6-1 compared to Ler eds 1-2and additionally the DMR6 transcript. FIG. 10B shows elevated genetranscripts of six defence-associated genes in dmr6-2 versus Ws-4. ACTreflects the number of additional PCR amplification cycles required toreach the level of ACTIN2 transcripts. A lower ACT value indicates ahigher transcript level.

FIG. 11 shows the nucleotide sequence (SEQ ID NO: 107) of the 3 kbregion upstream of the start codon of the DMR6 gene (at5g24530) ofArabidopsis thaliana, including the promoter and 5′-UTR (underlined).

FIG. 12 shows the nucleotide (SEQ ID NO: 95) and derived amino acidsequence (SEQ ID NO: 96) of the DMR6 ortholog of Solanum lycopersicum,respectively.

FIG. 13 shows the nucleotide (SEQ ID NO: 97) and derived amino acidsequence (SEQ ID NO: 98) of the DMR6 ortholog of Nicotiana benthamiana,respectively.

FIG. 14 shows complementation of Arabidopsis thaliana dmr6-1 with DMR6derived from Cucumis sativa (Cs), Spinacia oleracea (Si), Lactuca sativa(Ls) and Solanum lycopersicum (So).

FIG. 15 shows a box plot of the whole plant downy mildew test resultsobtained 8 days post infection (dpi) from a segregating F2 population ofrapid cycling Brassica oleracea (FastPlant®,https://fastplants.org/origin/) containing the mutant alleles bodmr6-1-1and bodmr6-2. The y-axis lists the genotypes, and provides the number ofplants tested: wild type (“AA BB”), 27 plants; and homozygous doublemutant for mutant alleles bodmr6-1-1 and bodmr6-2 (“aa bb”), 13 plants.For all genotypes, “+” is the sample mean, “º” is a single data point,and the box reflects the variation within each genotype. The diseasescore shown on the x-axis ranges from 0 (susceptible) to 9 (resistant)(see Table 6).

FIG. 16 shows the phenotypic difference between homozygous double mutantfor mutant alleles bodmr6-1-1 and bodmr6-2 (“aa bb”) and wild type (“AABB”) rapid cycling Brassica oleracea (FastPlant®,https://fastplants.org/origin/) plants inoculated with downy mildew(Hyaloperonospora parasitica/Hyaloperonospora brassicae) at 8 dpi.

FIGS. 17A-17C show box plots of the leaf disc downy mildew test resultsobtained 9 dpi from three different segregating F2 populations of rapidcycling Brassica oleracea (FastPlant®, https://fastplants.org/origin/).FIG. 17A shows the results from F2 pop. 1 containing the mutant allelesbodmr6-1-1 and bodmr6-2. The y-axis lists the genotypes, and providesthe number of plants tested: wild type (“AA BB”), 5 plants; andhomozygous double mutant for mutant alleles bodmr6-1-1 and bodmr6-2 (“aabb”), 15 plants. FIG. 17B shows the results from F2 pop. 2 containingthe mutant alleles bodmr6-1-2 and bodmr6-2. The y-axis lists thegenotypes, and provides the number of plants tested: wild type (“AABB”), 8 plants; and homozygous double mutant for mutant allelesbodmr6-1-2 and bodmr6-2 (“aa bb”), 15 plants. FIG. 17C shows the resultsfrom F2 pop. 3 containing the mutant alleles bodmr6-1-3 and bodmr6-2.The y-axis lists the genotypes, and provides the number of plantstested: wild type (“AA BB”), 5 plants; and homozygous double mutant formutant alleles bodmr6-1-2 and bodmr6-2 (“aa bb”), 10 plants. For FIGS.17A-17C, “+” is the sample mean, “0” is a single data point, the boxreflects the variation within each genotype, and the disease score shownon the x-axis ranges from 0 (susceptible) to 9 (resistant) (see Table6).

FIGS. 18A-18D show exemplary images of infected plants from aXanthomonas campestris campestris (Xcc) test with different diseaseseverity scores. FIG. 18A shows a plant with disease severity score 1(i.e., local symptom=all leaves are more than 50% infected; systemicsymptom=plant is dead; see Table 9). FIG. 18B shows a plant with diseaseseverity score 3 (i.e., local symptom=all leaves are less than 50%infected; systemic symptom=all new leaves are infected; see Table 9).FIG. 18C shows a plant with disease severity score 5 (i.e., localsymptom=1 or 2 leaves infected with V-shaped lesions; systemic symptom=1or 2 new leaves infected with a V-shaped lesion; see Table 9). FIG. 18Dshows a plant with disease severity score 7 (i.e., local symptom=noclear infection, but some leaves show spots; systemic symptom=no clearinfection, but older leaves show some symptoms; see Table 9). For FIGS.18A-18D, the red ring marks the youngest leaf developed at the time ofinfection, and symptoms observed above the red ring are consideredsystemic, while symptoms observed below the red ring are consideredlocal.

FIGS. 19A-19D show box plots of results from Xcc tests using differentXcc strains (Xcc 1 or Xcc 4) to infect DMR6-1 and DMR6-2 double knockoutand wild type plants. FIG. 19A shows scores of local infection from anXcc test that used Xcc race 1 (Xcc 1) to test resistant control plants(R1, 26 plants; R2, 30 plants), susceptible control plants (KT38, 30plants), DMR6-1 and DMR6-2 double knockout plants (GE, 38 plants), andwild type plants processed with the same protocol as the GE plants(i.e., plants regenerated from protoplasts that did not contain thedouble mutation; GE.WT, 37 plants). FIG. 19B shows scores of systemicinfection from an Xcc test that used Xcc 1 to test resistant controlplants (R1, 26 plants; R2, 30 plants), susceptible control plants (KT38,30 plants), DMR6-1 and DMR6-2 double knockout plants (GE, 38 plants),and wild type plants processed with the same protocol as the GE plants(i.e., plants regenerated from protoplasts that did not contain thedouble mutation; GE.WT, 37 plants). FIG. 19C shows scores of localinfection from an Xcc test that used Xcc race 4 (Xcc 4) to testresistant control plants (R1, 5 plants; R2, 4 plants), susceptiblecontrol plants (KT38, 6 plants), DMR6-1 and DMR6-2 double knockoutplants (GE, 33 plants), and wild type plants processed with the sameprotocol as the GE plants (i.e., plants regenerated from protoplaststhat did not contain the double mutation; GE.WT, 27 plants). FIG. 19Dshows scores of systemic infection from an Xcc test that used Xcc 4 totest resistant control plants (R1, 5 plants; R2, 4 plants), susceptiblecontrol plants (KT38, 6 plants), DMR6-1 and DMR6-2 double knockoutplants (GE, 33 plants), and wild type plants processed with the sameprotocol as the GE plants (i.e., plants regenerated from protoplaststhat did not contain the double mutation; GE.WT, 27 plants). For FIGS.19A-19D, “+” is the sample mean, “º” is a single data point, the boxreflects the variation within each genotype, the results are thecombined results from two independent tests, and the disease score shownon the x-axis ranges from 0 (susceptible) to 9 (resistant).

FIG. 20 shows an exemplary image of the systemic resistance phenotype ofa DMR6-1 and DMR6-2 double knockout plant (GE; on right) and a wild typeplant processed with the same protocol as the GE plant (i.e., a plantregenerated from a protoplast that did not contain the double mutation;GE.WT, on left). Pictures were taken four weeks after infection withXcc.

FIG. 21 shows a box plot of whole plant downy mildew test resultsobtained 8 dpi from DMR6-1 and DMR6-2 double knockout plants (GE, 22plant), and wild type plants processed with the same protocol as the GEplants (i.e., plants regenerated from protoplasts that did not containthe double mutation; WT, 18 plants). “+” is the sample mean, “º” is asingle data point, the box reflects the variation within each genotype,the results are the combined results from two independent tests, and thedisease score shown on the x-axis ranges from 2 (susceptible) to 9(resistant).

FIGS. 22A-22F show the alignment of the wild type DMR6-1 nucleotidesequence (BoDMR6_WT_scf108=SEQ ID NO: 114; top row) with mutated DMR6-1nucleotide sequences (BoDMR6_C181T_scf=SEQ ID NO: 128;BoDMR6_C691T_scf=SEQ ID NO: 129; BoDMR6_G714A_scf=SEQ ID NO: 130;BoDMR6_−5 bp_scf1=SEQ ID NO: 137; bottom four rows).

FIGS. 23A-23E show the alignment of the wild type DMR6-2 nucleotidesequence (BoDMR6_WT_scf155=SEQ ID NO: 115; top row) with mutated DMR6-2nucleotide sequences (BoDMR6_G368A_scf=SEQ ID NO: 131;BoDMR6_-lbp_scf1=SEQ ID NO: 138; bottom two rows).

FIGS. 24A-24B show the alignment of the wild type DMR6-1 polypeptidesequence (BoDMR6_WT_scf108=SEQ ID NO: 112; top row) with mutated DMR6-1polypeptide sequences (BoDMR6_C181T_scf=SEQ ID NO: 141;BoDMR6_C691T_scf=SEQ ID NO: 142; BoDMR6_G714A_scf=SEQ ID NO: 143;BoDMR6_−5 bp_scf1=SEQ ID NO: 139; bottom four rows).

FIGS. 25A-25B show the alignment of the wild type DMR6-2 polypeptidesequence (BoDMR6_WT_scf155=SEQ ID NO: 113; top row) with mutated DMR6-2nucleotide sequences (BoDMR6_G368A_scf=SEQ ID NO: 144;BoDMR6_-lbp_scf1=SEQ ID NO: 140; bottom two rows).

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows orthologous DMR6 sequences (described in Table 1) that havebeen identified in publicly available databases and obtained by PCRamplification on cDNA and subsequent sequencing. After orthologous DMR6sequences are identified, the complete nucleotide sequence of theregulatory and coding sequence of the gene is identified by standardmolecular biological techniques. For this, genomic libraries of theplant species are screened by DNA hybridization or PCR with probes orprimers derived from a known DMR6 gene to identify the genomic clonescontaining the DMR6 gene. Alternatively, advanced PCR methods, such asRNA ligase-mediated RACE (RLM-RACE), can be used to directly amplifygene and cDNA sequences from genomic DNA or reverse-transcribed mRNA.DNA sequencing subsequently results in the characterization of thecomplete gene or coding sequence.

Once the DNA sequence of the gene is known this information is used toprepare the means to modulate the expression of the DMR6 gene.

To achieve a reduced DMR6 protein level, the expression of the DMR6 genecan be down-regulated or the enzymatic activity of the DMR6 protein canbe reduced by amino acid substitutions resulting from nucleotide changesin the DMR6 coding sequence.

In a particular embodiment of the invention, downregulation of DMR6 geneexpression is achieved by gene-silencing using RNAi. For this,transgenic plants are generated expressing a DMR6 anti-sense construct,an optimized micro-RNA construct, an inverted repeat construct, or acombined sense-anti-sense construct, so as to generate dsRNAcorresponding to DMR6 that leads to gene silencing.

In an alternative embodiment, one or more regulators of the DMR6 geneare downregulated (in case of transcriptional activators) by RNAi.

In another embodiment regulators are upregulated (in case of repressorproteins) by transgenic overexpression. Overexpression is achieved in aparticular embodiment by expressing repressor proteins of the DMR6 genefrom a strong promoter, e.g. the 35S promoter that is commonly used inplant biotechnology.

The downregulation of the DMR6 gene can also be achieved by mutagenesisof the regulatory elements in the promoter, terminator region, orpotential introns. Mutations in the DMR6 coding sequence in many casesleads to amino acid substitutions or premature stop codons thatnegatively affect the expression or activity of the encoded DMR6protein. In a particular embodiment of the invention, the mutations inthe DMR6 coding sequence are the introduction of a premature stop codon.In a further embodiment, mutations in the two cabbage DMR6 genes (i.e.,DMR6-1 and DMR6-2) introduce a premature stop codon into each of thecabbage DMR6 genes.

These mutations are induced in plants by using mutagenic chemicals suchas ethyl methane sulfonate (EMS), by irradiation of plant material withgamma rays or fast neutrons, or by other means. The resulting nucleotidechanges are random, but in a large collection of mutagenized plants themutations in the DMR6 gene can be readily identified by using theTILLING (Targeting Induced Local Lesions IN Genomes) method (McCallum etal. (2000) Targeted screening for induced mutations. Nat. Biotechnol.18, 455-457, and Henikoff et al. (2004) TILLING. Traditional mutagenesismeets functional genomics. Plant Physiol. 135, 630-636). The principleof this method is based on the PCR amplification of the gene of interestfrom genomic DNA of a large collection of mutagenized plants in the M2generation. By DNA sequencing or by looking for point mutations using asingle-strand specific nuclease, such as the CEL-I nuclease (Till et al.(2004) Mismatch cleavage by single-strand specific nucleases. NucleicAcids Res. 32, 2632-2641) the individual plants that have a mutation inthe gene of interest are identified.

By screening many plants, a large collection of mutant alleles isobtained, each giving a different effect on gene expression or enzymeactivity. The gene expression or protein levels can for example betested by analysis of DMR6 transcript levels (e.g. by RT-PCR) or byquantification of DMR6 protein levels with antibodies.

Plants with the desired reduced DMR6 level or DMR6 expression are thenback-crossed or crossed to other breeding lines to transfer only thedesired new allele into the background of the crop wanted.

The invention further relates to mutated DMR6 genes. In a particularembodiment, the invention relates to dmr6 alleles with premature stopcodons, such as the dmr6-1 allele and the dmr6-2 allele.

In another embodiment, the invention relates to mutated versions of theDMR6 genes of Lactuca sativa, Cucumis sativus, and Spinacia oleracea asshown in FIGS. 3-5.

The present invention demonstrates that plants having no or a reducedlevel of functional DMR6 gene product show resistance to pathogens, inparticular of oomycete and fungal origin. With such knowledge theskilled person can identify so far unknown natural variants of a givenplant species that have variants of the DMR6 gene that lead to a reducedlevel or absence of a functional DMR6 protein, or mutated versions ofthe DMR6 protein, and to use these natural variants according to theinvention.

The present invention further relates to the use of a DMR6 promoter forproviding disease resistance into plants, i.e. for providing plants witha resistance to a pathogen of viral, bacterial, fungal or oomyceteorigin. According to the present invention, the transcriptionalup-regulation of DMR6 in response to pathogen infection has beendemonstrated. Both transcript analysis as well as promoter DMR6-reporterlines support this finding (see Example 1, below). Thepathogen-inducible DMR6 promoter according to the invention thus isparticularly useful to control the expression of inducible systems thatlead to disease resistance in plants.

One example of such inducible system that leads to disease resistance inplants, and in which the DMR6 promoter according to the presentinvention may be effective, has e.g. been described in WO 99/45125,wherein an antisense nucleotide sequence for a gene involved in theregulation of the C-5 porphyrin metabolic pathway is operably linked toa pathogen-inducible promoter and used to transform plant cells.Expression of the antisense nucleotide sequence in response to thepathogen effectively disrupts porphyrin metabolism of the transformedplant cell, and development of a localized lesion wherein the spread ofthe pathogen is contained. WO 96/36697 also discloses inducible systemsleading to disease resistance in plants, wherein an inducible promotercontrols the expression of a protein capable of evoking thehypersensitivity response in a plant. EP 0474857 furthermore discloses amethod for the induction of pathogen resistance in plants, comprisingtransforming plants with polynucleotide sequences encoding a pair ofpathogen-derived-avirulence-gene/plant-derived-resistance gene, whereinthe expression of one of or both the elicitor peptide and the resistancegene is regulated by a pathogen inducible promoter. Further examples ofinducible systems leading to resistance to pathogens in plants have beendescribed in e.g. WO 98/32325.

In a particular preferred embodiment, the present invention relates to amethod of providing disease resistance in a plant, comprisingtransforming a plant cell with a DNA construct comprising at least oneexpressible nucleic acid which is operably linked to apathogen-inducible promoter that is operable within a plant cell, andregenerating transformed plants from said plant cells, wherein thepathogen-inducible promoter is a DMR6 promoter, and wherein theexpression of the expressible nucleic acid confers disease resistance tothe transgenic plant.

The invention also relates to disease resistance plants, obtainable bysaid method, as well as to plant tissue, and seeds obtained from saidplants.

The invention in particular relates to plants, which are resistant to apathogen of viral, bacterial, fungal or oomycete origin, wherein theplant comprises in its genome a DNA construct, comprising at least oneexpressible nucleic acid which is operably linked to apathogen-inducible promoter, wherein the pathogen-inducible promoter isa DMR6 promoter.

The present invention also relates to the DNA construct per se,comprising at least one expressible nucleic acid which is operablylinked to a pathogen-inducible promoter, wherein the pathogen-induciblepromoter is a DMR6 promoter. The construct of the invention can be usedto transform plant cells which may be regenerated into transformedplants. Furthermore, transformed plant tissue and seed may be obtained.Suitable methods for introducing the construct of the invention intoplant cells are known to the skilled person.

According to the invention, by “operably linked” is meant that apromoter and an expressible nucleic acid, e.g. a gene, are connected insuch way as to permit initiation of transcription of the expressiblenucleic acid (e.g. gene) by the promoter.

By “expressible nucleic acid” is meant a nucleic acid (e.g. a gene, orpart of a gene) that can be expressed in the cell, i.e., that can betranscribed into mRNA, and eventually may be translated into a protein.The expressible nucleic acid may be genomic DNA, cDNA, or chemicallysynthesized DNA or any combination thereof.

According to the present invention, a DNA construct comprises allnecessary nucleic acid elements which permit expression (i.e.transcription) of a particular nucleic acid in a cell. Typically, theconstruct includes an expressible nucleic acid, i.e. a nucleic acid tobe transcribed, and a promoter. The construct can suitably beincorporated into e.g. a plasmid or vector.

The expressible nucleic acid preferably is a gene involved in a plantdefence response, e.g. a gene associated with the hypersensitivityresponse of a plant. In the hypersensitivity response (HR) of a plant,the site in the plant where the pathogen invades undergoes localizedcell death by the induced expression of a suicide mechanism thattriggers said localized cell death in response to pathogens. In thisway, only a few plant cells are sacrificed and the spread of thepathogen is effectively arrested. Examples of said genes involved in aplant defence response are the regulatory protein NPR1/NIM1 (Friedrichet al., Mol. Plant Microbe Interact. 14(9): 1114-1124, 2001) and thetranscription factor MYB30 (Vailleau et al., Proc. Natl. Acad. Sci. USA99(15): 10179-10184, 2002).

In a particular embodiment, the expressible nucleic acid encodes anautologous or heterologous polypeptide capable of conferringdisease-resistance to a plant. By “autologous polypeptide” is meant anypolypeptide that is expressed in a transformed plant cell from a genethat naturally occurs in the transformed plant cell. By “heterologouspolypeptide” is meant any polypeptide that is expressed in a transformedplant cell from a gene that is partly or entirely foreign (i.e. does notnaturally occur in) to the transformed plant cell. Examples of suchpolypeptides are the mammalian Bax protein, which encodes apro-apoptotic protein and results in cell death in plants (Lacomme andSanta Cruz, Proc. Natl. Acad. Sci. USA 96(14): 7956-61, 1999) and fungalchitinases (de las Mercedes Dana et al., Plant Physiol. 142(2): 722-730,2006).

Preferably, the DMR6 promoter is the Arabidopsis DMR6 promoter. The DMR6promoter comprises a region of 3000 bp that is upstream of theArabidopsis DMR6 coding sequence (ATG start codon) and includes the5′UTR. Preferably the DMR6 promoter comprises a nucleotide sequence asdefined in FIG. 11, and/or any functional fragment thereof, i.e. anyfragment (or part) of said sequence which still is capable of initiatingtranscription of the expressible nucleic acid(s) to which it is operablylinked, and/or natural variants thereof, i.e. natural variants of thispromoter which may contain small polymorphisms, but which are generallyat least 90% identical.

In a further preferred embodiment, the DMR6 promoter is an orthologousDMR6 promoter, i.e. a promoter of an orthologous DMR6 gene. Methods foridentifying DMR6 orthologs have been described in Example 2 below. Oncethe DMR6 orthologs have been identified, the skilled person will be ableto isolate the respective promoter of said orthologs, using standardmolecular biological techniques.

According to the present invention, the DMR6 promoter has been shown tobe strongly pathogen-induced, and the DMR6 promoter is not highlyexpressed in other non-infected tissues. Thus, it is a very suitablepromoter for use in inducible systems for providing resistance topathogens of viral, bacterial, fungal or oomycete origin in plants.Examples of specific pathogens and plants for which the induciblesystem, using the DMR6 promoter of the present invention, suitably canbe used, have been given above.

In a particular embodiment, downregulation of DMR6 gene expression isachieved by gene silencing, RNA interference (RNAi), virus-induced genesilencing (VIGS), small RNA-mediated post-transcriptional genesilencing, transcription activator-like effector nuclease (TALEN) geneediting techniques, clustered Regularly Interspaced Short PalindromicRepeat (CRISPR/Cas9) gene editing techniques, and/or zinc-fingernuclease (ZFN) gene editing techniques. For this, transgenic plants aregenerated expressing one or more constructs targeting DMR6. Theseconstructs may include, without limitation, an anti-sense construct, anoptimized small-RNA construct, an inverted repeat construct, a targetingconstruct, a guide RNA construct, a construct encoding a targetingprotein, and/or a combined sense-anti-sense construct, and may work inconjunction with a nuclease, an endonuclease, and/or an enzyme, so as todownregulate DMR6 gene expression.

In an alternative embodiment, one or more regulators of the DMR6 geneare downregulated (in case of transcriptional activators) by RNAinterference (RNAi), virus-induced gene silencing (VIGS), smallRNA-mediated post-transcriptional gene silencing, transcriptionactivator-like effector nuclease (TALEN) gene editing techniques,clustered Regularly Interspaced Short Palindromic Repeat (CRISPR/Cas9)gene editing techniques, and/or zinc-finger nuclease (ZFN) gene editingtechniques.

Brassica Plants of the Present Disclosure

Accordingly, certain aspects of the present disclosure relate to amutant Brassica plant, wherein the plant has a reduced activity or areduced level of a DMR6-1 polypeptide as compared to a correspondingwild type Brassica plant, and wherein the mutant Brassica plant has atleast one non-natural mutation introduced into the DMR6-1 gene; andwherein the mutant Brassica plant has a reduced activity or a reducedlevel of a DMR6-2 polypeptide as compared to a corresponding wild typeBrassica plant, and wherein the mutant Brassica plant has at least onenon-natural mutation introduced into the DMR6-2 gene. In someembodiments, the plant exhibits resistance selected from the group ofresistance to Hyaloperonospora parasitica/Hyaloperonospora brassicae,intermediate resistance to Xanthomonas campestris campestris, or anycombination thereof. In some embodiments, the DMR6-1 polypeptide isselected from the group of a polypeptide with at least 85% sequenceidentity, at least 88% sequence identity, at least 89% sequenceidentity, at least 90% sequence identity, at least 91% sequenceidentity, at least 92% sequence identity, at least 93% sequenceidentity, at least 94% sequence identity, at least 95% sequenceidentity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, or at least 99% sequenceidentity to SEQ ID NO: 112; and wherein the DMR6-2 polypeptide is apolypeptide selected from the group of a protein with at least 85%sequence identity, at least 88% sequence identity, at least 89% sequenceidentity, at least 90% sequence identity, at least 91% sequenceidentity, at least 92% sequence identity, at least 93% sequenceidentity, at least 94% sequence identity, at least 95% sequenceidentity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, or at least 99% sequenceidentity to SEQ ID NO: 113. In some embodiments that may be combinedwith any of the above embodiments, the DMR6-1 gene is selected from thegroup of a nucleotide with at least 85% sequence identity, at least 88%sequence identity, at least 89% sequence identity, at least 90% sequenceidentity, at least 91% sequence identity, at least 92% sequenceidentity, at least 93% sequence identity, at least 94% sequenceidentity, at least 95% sequence identity, at least 96% sequenceidentity, at least 97% sequence identity, at least 98% sequenceidentity, or at least 99% sequence identity to SEQ ID NO: 114; andwherein the DMR6-2 gene is selected from the group of a nucleotide with85% sequence identity, at least 88% sequence identity, at least 89%sequence identity, at least 90% sequence identity, at least 91% sequenceidentity, at least 92% sequence identity, at least 93% sequenceidentity, at least 94% sequence identity, at least 95% sequenceidentity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, or at least 99% sequenceidentity to SEQ ID NO: 115. In some embodiments that may be combinedwith any of the above embodiments, the non-natural mutation in DMR6-1 isa premature stop codon, and the non-natural mutation in DMR6-2 is apremature stop codon.

In some embodiments, the at least one non-natural mutation introducedinto the DMR6-1 gene reduces an activity or a level of a DMR6-1polypeptide as compared to a corresponding wild type Brassica plant; andthe at least one non-natural mutation introduced into the DMR6-2 genereduces an activity or a level of a DMR6-2 polypeptide as compared to acorresponding wild type Brassica plant. In some embodiments, the DMR6-1polypeptide is selected from the group of a polypeptide with 85%sequence identity, at least 88% sequence identity, at least 89% sequenceidentity, at least 90% sequence identity, at least 91% sequenceidentity, at least 92% sequence identity, at least 93% sequenceidentity, at least 94% sequence identity, at least 95% sequenceidentity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, or at least 99% sequenceidentity to SEQ ID NO: 112; and wherein the DMR6-2 polypeptide is apolypeptide selected from the group of a protein with at least 85%sequence identity, at least 88% sequence identity, at least 89% sequenceidentity, at least 90% sequence identity, at least 91% sequenceidentity, at least 92% sequence identity, at least 93% sequenceidentity, at least 94% sequence identity, at least 95% sequenceidentity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, or at least 99% sequenceidentity to SEQ ID NO: 113. In some embodiments that may be combinedwith any of the above embodiments, the DMR6-1 gene is selected from thegroup of a nucleotide with at least 85% sequence identity, at least 88%sequence identity, at least 89% sequence identity, at least 90% sequenceidentity, at least 91% sequence identity, at least 92% sequenceidentity, at least 93% sequence identity, at least 94% sequenceidentity, at least 95% sequence identity, at least 96% sequenceidentity, at least 97% sequence identity, at least 98% sequenceidentity, or at least 99% sequence identity to SEQ ID NO: 114; andwherein the DMR6-2 gene is selected from the group of a nucleotide with85% sequence identity, at least 88% sequence identity, at least 89%sequence identity, at least 90% sequence identity, at least 91% sequenceidentity, at least 92% sequence identity, at least 93% sequenceidentity, at least 94% sequence identity, at least 95% sequenceidentity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, or at least 99% sequenceidentity to SEQ ID NO: 115.

In some embodiments, the at least one non-natural mutation that reducesthe activity or level of the DMR6-1 protein is selected from the groupof a premature stop codon introduced into the DMR6-1 coding sequence, aframeshift mutation introduced into the DMR6-1 coding sequence, aninsertion introduced into the DMR6-1 coding sequence, a deletion of apart or a whole of the DMR6-1 coding sequence, an altered amino acid ina conserved domain of the DMR6-1 protein, a modified upstream sequence,a mutated promoter element, or an activated repressor element; and theat least one non-natural mutation that reduces the activity or level ofthe DMR6-2 protein is selected from the group of a premature stop codonintroduced into the DMR6-2 coding sequence, a frameshift mutationintroduced into the DMR6-2 coding sequence, an insertion introduced intothe DMR6-2 coding sequence, a deletion of a part or a whole of theDMR6-2 coding sequence, an altered amino acid in a conserved domain ofthe DMR6-2 protein, a modified upstream sequence, a mutated promoterelement, or an activated repressor element.

In one embodiment, the plant of the present disclosure may be obtainedthrough introduction of a premature stop codon into Brassica DMR6-1 andDMR6-2 genes. In some embodiments, the introduction of a premature stopcodon may be through a single nucleotide change, a single nucleotidedeletion, the deletion of five nucleotides, the deletion of nucleotidessuch that a frameshift mutation is produced, or the insertion ofnucleotides such that a frameshift mutation is produced. In a particularembodiment, the Cytosine (C) is replaced with a Thymine (T) at aposition of DMR6-1 corresponding to nucleotide 181 of the referencesequence SEQ ID NO: 114 (e.g., producing SEQ ID NO: 128), resulting in achange from Arginine (R) to a stop codon (e.g., producing SEQ ID NO:141). In another particular embodiment, the C is replaced with a T at aposition of DMR6-1 corresponding to nucleotide 691 of the referencesequence SEQ ID NO: 114 (e.g., producing SEQ ID NO: 129), resulting in achange from Glutamine (Q) to a stop codon (e.g., producing SEQ ID NO:142). In a further particular embodiment, the Guanine (G) is replacedwith an Adenine (A) at a position of DMR6-1 corresponding to nucleotide714 of the reference sequence of the reference sequence SEQ ID NO: 114(e.g., producing SEQ ID NO: 130) resulting in a change from Tryptophan(W) to a stop codon (e.g., producing SEQ ID NO: 143). In yet anotherparticular embodiment, five nucleotides (e.g., CCTGA) are deletedbeginning at a position of DMR6-1 corresponding to nucleotide 601 of thereference sequence SEQ ID NO: 114 (e.g., producing SEQ ID NO: 137),producing a frameshift mutation and a premature stop codon (e.g.,producing SEQ ID NO: 139). In an additional particular embodiment, the Gis replaced with an A at a position of DMR6-2 corresponding tonucleotide 368 of the reference sequence SEQ ID NO: 115 (e.g., producingSEQ ID NO: 131) resulting in a change from W to a stop codon (e.g.,producing SEQ ID NO: 144). In a further particular embodiment, thenucleotide is deleted at a position of DMR6-2 corresponding tonucleotide 600 of the reference sequence SEQ ID NO: 115 (e.g., producingSEQ ID NO: 138), producing a frameshift mutation and a premature stopcodon (e.g., producing SEQ ID NO: 140). In some embodiments, thenon-natural mutation introduced into the DMR6-1 gene is selected fromthe group of a C to T mutation at a position corresponding to nucleotide181 of reference sequence SEQ ID NO: 114, a C to T mutation at aposition corresponding to nucleotide 691 of reference sequence SEQ IDNO: 114, a G to A mutation at a position corresponding to nucleotide 714of reference sequence SEQ ID NO: 114, or a deletion of five nucleotidesstarting at a position corresponding to nucleotide 601 of referencesequence SEQ ID NO: 114; and the non-natural mutation introduced intothe DMR6-2 gene is selected from the group of a G to A mutation at aposition corresponding to nucleotide 368 of reference sequence SEQ IDNO: 115, or a deletion of the nucleotide at a position corresponding tonucleotide 600 of reference sequence SEQ ID NO: 115.

Without wishing to be limited by theory, in some embodiments, thepremature stop codon is located before or within the essential ironbinding residue conserved in all 20G oxygenases such as DMR6 (Wilmouthet al. (2002), Structure, 10:93-103). Without wishing to be limited bytheory, in another embodiment, the plant of the present disclosure maybe obtained through introduction of at least one amino acid change inthe essential iron binding residue of DMR6-1 and the introduction of atleast one amino acid change in the essential iron binding residue ofDMR6-2. Without wishing to be limited by theory, in a furtherembodiment, the plant of the present disclosure may be obtained throughintroduction of at least one mutation in DMR6-1 and at least onemutation in DMR6-2 resulting in altered DMR6-1 function and alteredDMR6-2 function that can be detected as an increase of salicylic acid(SA) accumulation.

In some embodiments, the non-natural mutation introduced into the DMR6-1gene is selected from the group of a C to T mutation at position 181corresponding to the reference sequence SEQ ID NO: 128, a C to Tmutation at position 691 corresponding to the reference sequence SEQ IDNO: 129, a G to A mutation at position 714 corresponding to thereference sequence SEQ ID NO: 130, or deletion of CCTGA at position 601corresponding to reference sequence SEQ ID NO: 137; and wherein thenon-natural mutation introduced into the DMR6-2 gene is selected fromthe group of a G to A mutation at position 368 corresponding toreference sequence SEQ ID NO: 131, or deletion of T at position 600corresponding to reference sequence SEQ ID NO: 138.

In some embodiments that may be combined with any of the aboveembodiments, the Brassica plant is a plant of the family Brassicaceae(i.e., Cruciferae). In some embodiments, the plant is Brassica oleracea.In some embodiments, the plant is a Brassica oleracea cultivar selectedfrom the group of cabbage, kohlrabi, kale, Brussels sprouts, collardgreens, broccoli, Romanesco broccoli, or cauliflower.

In some embodiments, the present disclosure relates to a seed, tissue,or plant part of the Brassica plant of any of the above embodiments thatincludes a reduced activity or a reduced level of a DMR6-1 polypeptideas compared to a corresponding wild type Brassica plant and a reducedactivity or a reduced level of a DMR6-2 polypeptide as compared to acorresponding wild type Brassica plant, and wherein the seed, tissue, orplant part includes at least one non-natural mutation in the DMR6-1 geneand at least one non-natural mutation in the DMR6-2 gene.

In some embodiments of any of the above embodiments, the presentdisclosure relates to a plant part, wherein the plant part is a leaf, astem, a root, a flower, a seed, a fruit, a cell, or a portion thereof.In some embodiments, the plant part is a leaf.

In some aspects, the present disclosure relates to a pollen grain or anovule of the plant of any of the above embodiments. In some aspects, thepresent disclosure relates to a protoplast produced from the plant ofany of the above embodiments. In some aspects, the present disclosurerelates to a tissue culture produced from protoplasts or cells from theplant of any of the above embodiments, wherein the cells or protoplastsare produced from a plant part selected from the group of leaf, anther,pistil, stem, petiole, root, root primordia, root tip, fruit, seed,flower, cotyledon, hypocotyl, embryo, or meristematic cell. In someaspects, the present disclosure relates to a plant seed produced fromthe plant of any of the above embodiments.

In order to determine whether a plant is a plant of the presentdisclosure, and therefore whether said plant has the same alleles asplants of the present disclosure, the phenotype of the plant can becompared with the phenotype of a known plant of the present disclosure.In one embodiment, the phenotype can be assessed by, for example, thesusceptibility to downy mildew in a whole plant test assay.

In the whole plant assay, plant with the 1^(st) leaf pair are inoculatedwith 2×10⁴ spores (20,000 spores/mL) of a downy mildew isolate (e.g.,Hyaloperonospora parasitica/Hyaloperonospora brassicae Y113). Inoculatedplants are kept in a climate cell kept at 15° C. with a day-night cycleof 16 hours light-8 hours dark (16:8 cycle). Then, the whole plant downymildew test results are scored 8 to 9 days after inoculation using thescoring scale in Table 6.

In another embodiment, the phenotype can be assessed by measuringsalicylic acid (SA) accumulation in the plant leaves. A plant of thepresent disclosure will have increased SA accumulation as compared to aWT plant. Methods of measuring SA accumulation are described inZeilmaker et al. (2015), The Plant Journal, 81: 210-222 and Zhang et al.(2017), Plant Physiology, DOI:10.1104/pp. 17.00695.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques known in the artthat are available for the analysis, comparison and characterization ofplant genotype. Such techniques include, without limitation, IsozymeElectrophoresis, Restriction Fragment Length Polymorphisms (RFLPs),Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily PrimedPolymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARs), AmplifiedFragment Length polymorphisms (AFLPs), Simple Sequence Repeats (SSRs,which are also referred to as Microsatellites), and Single NucleotidePolymorphisms (SNPs). By using these techniques, it is possible toassess the presence of the alleles, genes, and/or loci involved in thedowny mildew resistance phenotype of the plants of the presentdisclosure.

Methods for Obtaining Brassica Plants of the Present Disclosure

Further aspects of the present disclosure relate to methods forobtaining a mutant Brassica plant including: introducing at least onenon-natural mutation into the DMR6-1 gene and introducing at least onenon-natural mutation into the DMR6-2 gene to produce a mutant Brassicaplant with a reduced activity or a reduced level of a DMR6-1 polypeptideas compared to a corresponding wild type Brassica plant and a reducedactivity or a reduced level of a DMR6-2 polypeptide as compared to acorresponding wild type Brassica plant. In some embodiments, the mutantBrassica plant exhibits resistance selected from the group of resistanceto Hyaloperonospora parasitica/Hyaloperonospora brassicae, intermediateresistance to Xanthomonas campestris campestris, or any combinationthereof. In some embodiments, the non-natural mutation is achieved by amutagenic treatment (e.g., EMS), a radiation treatment, or a geneediting technique. In some embodiments, the gene editing technique isselected from the group of transcription activator-like effectornuclease (TALEN) gene editing techniques, clustered RegularlyInterspaced Short Palindromic Repeat (CRISPR/Cas9) gene editingtechniques, or zinc-finger nuclease (ZFN) gene editing techniques.

In some embodiments, the mutant Brassica plant produced from the abovemethods includes at least one non-natural mutation in the DMR6-1 geneand at least one non-natural mutation in the DMR6-2 gene, wherein theplant further comprises a reduced activity or a reduced level of aDMR6-1 polypeptide as compared to a corresponding wild type Brassicaplant and a reduced activity or a reduced level of a DMR6-2 polypeptideas compared to a corresponding wild type Brassica plant, and wherein theplant exhibits resistance selected from the group of resistance toHyaloperonospora parasitica/Hyaloperonospora brassicae, intermediateresistance to Xanthomonas campestris campestris, or any combinationthereof. In some embodiments, the mutant Brassica plant produced fromthe above methods includes least one non-natural mutation in the DMR6-1gene that reduces an activity or a level of a DMR6-1 polypeptide ascompared to a corresponding wild type Brassica plant; wherein the plantincludes at least one non-natural mutation in the DMR6-2 gene thatreduces an activity or a level of a DMR6-2 polypeptide as compared to acorresponding wild type Brassica plant; and wherein the plant exhibitsresistance selected from the group of resistance to Hyaloperonosporaparasitica/Hyaloperonospora brassicae, intermediate resistance toXanthomonas campestris campestris, or any combination thereof. In someembodiments, the DMR6-1 polypeptide is selected from the group of apolypeptide with 85% sequence identity, at least 88% sequence identity,at least 89% sequence identity, at least 90% sequence identity, at least91% sequence identity, at least 92% sequence identity, at least 93%sequence identity, at least 94% sequence identity, at least 95% sequenceidentity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, or at least 99% sequenceidentity to SEQ ID NO: 112; and wherein the DMR6-2 polypeptide is apolypeptide selected from the group of a protein with 85% sequenceidentity, at least 88% sequence identity, at least 89% sequenceidentity, at least 90% sequence identity, at least 91% sequenceidentity, at least 92% sequence identity, at least 93% sequenceidentity, at least 94% sequence identity, at least 95% sequenceidentity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, or at least 99% sequenceidentity to SEQ ID NO: 113. In some embodiments, the DMR6-1 gene isselected from the group of a nucleotide with 85% sequence identity, atleast 88% sequence identity, at least 89% sequence identity, at least90% sequence identity, at least 91% sequence identity, at least 92%sequence identity, at least 93% sequence identity, at least 94% sequenceidentity, at least 95% sequence identity, at least 96% sequenceidentity, at least 97% sequence identity, at least 98% sequenceidentity, or at least 99% sequence identity to SEQ ID NO: 114; andwherein the DMR6-2 gene is selected from the group of a nucleotide with85% sequence identity, at least 88% sequence identity, at least 89%sequence identity, at least 90% sequence identity, at least 91% sequenceidentity, at least 92% sequence identity, at least 93% sequenceidentity, at least 94% sequence identity, at least 95% sequenceidentity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, or at least 99% sequenceidentity to SEQ ID NO: 115. In some embodiments that may be combinedwith any of the above embodiments, the Brassica plant produced from anyof the above methods is a plant of the family Brassicaceae (i.e.,Cruciferae). In some embodiments, the Brassica plant produced from anyof the above methods is Brassica oleracea. In some embodiments, theBrassica plant produced from any of the above methods is a Brassicaoleracea cultivar selected from the group of cabbage, kohlrabi, kale,Brussels sprouts, collard greens, broccoli, Romanesco broccoli, orcauliflower. In some embodiments, the present disclosure relates to aseed, tissue, or plant part of the Brassica plant produced from any ofthe above methods.

The present invention is illustrated in the following examples that arenot intended to limit the invention in any way. In the examplesreference is made to the figures described above.

Example 1 The Arabidopsis DMR6 (At5g24530) Gene is Required for DownyMildew Susceptibility Experimental Procedures

Hyaloperonospora parasitica Growth and Infection

H. parasitica isolate Waco9 was provided by Dr. M. Aarts (WUR,Wageningen, NL) and isolate Cala2 provided by Dr. E. Holub (Warwick HRI,Wellsboume, UK) and maintained on Arabidopsis Ws-0 and Ler,respectively. Inocula (400,000 spores per ml) were weekly transferred to10 day old healthy seedlings (Holub, E. B. et al., Mol. Plant MicrobeInteract. 7: 223-239, 1994) by use of a spray gun. Seedlings wereair-dried for approximately 45 minutes and incubated under a sealed lidat 100% relative humidity in a growth chamber at 16° C. with 9 hours oflight per day (100mE/m2/s). The sporulation levels were quantified 7days post inoculation (dpi) by counting the number of sporangiophoresper seedling, for at least 40 seedlings per tested line (FIG. 6A) or byisolating spores in water 5 dpi and determining the spore concentrationto give the number per mg leaf tissue (FIG. 6B).

Generation of Backcrossed Dmr6 Lines

The dmr6 mutants were back crossed twice (BC2) to the parental line Lereds 1-2 as well as Ler. The BC2 lines generated with Ler were selectedfor the presence of the wild type EDS1 gene by PCR analysis.

Cloning DMR6

Fine mapping of the dmr6 gene was done with PCR markers designed usingthe Cereon database to identify insertion and deletion (IND) differencesbetween Col-0 and Ler. The markers: IND_MOP9 in gene At5G24210;IND_K16H17 in gene At5G24420; IND_T4C12 in gene At5G24820; IND_T11H3 inbetween genes At5G24950_60 and IND_F21J6 in gene At5G25270 were used formapping (Table 2). An additional screen for new recombinants wasinitiated on 300 F2 plants resulting in eight F2 recombinant plantsbetween the two IND based markers IND_MOP9 and IND_T4C12, which flankeda region of 61 genes. Seven additional markers (M450-M590; Table 2)reduced the region to eighteen candidate genes for the dmr6 locus,between At5g24420 and At5g24590. Sequence analysis of At5g24530indicated a point mutation leading to a stop codon in exon 2 in thedmr6-1 mutant.

TABLE 2PCR primers for the markers used for the map-based cloning of DMR6.INDEL/ Name primer Gene enzyme Forward primer Reverse primer IND_MOP9At5G24210 tttgggaacagaaaaagt catattcaaaagggaaaatc tggaggt (SEQ IDccaga (SEQ ID NO: NO: 37) 38) IND_K16H17 At5g24420 tggggttgtggtttattctgtggccaatagtagttgatac ttgac (SEQ ID gcaaga (SEQ ID NO: 39) NO: 40)IND_T4C12 At5g24820 tctcgggtaagacacaa tattccaacttgcgacgtaggtcgagat (SEQ ID agcat (SEQ ID NO: NO: 41) 42) IND_T11H3 At5g24950-60cggcttttaacaacatattttc ccaattgggttatttacttc gat (SEQ ID NO:ca (SEQ ID NO: 44) 43) IND_F21J6 At5g25270 aacacatcaccaagatgcctctgccccaagaaatatt aatccaga (SEQ ID gagat (SEQ ID NO: NO: 45) 46) M450At5G24450 18 agctttgtatggtagtgcc gcggtatacgggggttaaa aatga (SEQ IDatcta (SEQ ID NO: NO: 47) 48) M490 At5g24490 TaqI atggccaaccactctttgtacaagcaagaagaacagc tac (SEQ ID NO: gaag (SEQ ID NO: 49) 50) M525At5g24520-30 TaqI gaaatttggttgttggcat tcaagatcttcatattctcattttatc (SEQ ID NO: cca (SEQ ID NO: 52 51) M545 At5G24540/50 41cagctgaagtatgtttcat cttgcaattgttgggactag cccttt (SEQ ID gtaa (SEQ ID NO:NO: 53) 54) M555 At5G24550/60 14 tcactaaccagtgaaaaa tatacagcgaatagcaaagggttgc (SEQ ID ccaag (SEQ ID NO: NO: 55) 56) M470 At5g24470 HphIccgcgagtgtaatatatct cagtttaacgcatgaagtgc ctcct (SEQ ID NO:tagt (SEQ ID NO: 57) 58) M590 At5g24590 PdmI gcatcatttgtaccgtacttagtggatactctgtccctg gagtc (SEQ ID aggt (SEQ ID NO: NO: 59 60)

Identification of a Dmr6 T-DNA Insertion Line

A second dmr6 allele was identified, 445D09 a FLAG T-DNA insertion linegenerated by INRA Versailles in the Ws-4 accession background. The T-DNAinsertion was confirmed by PCR using a primer designed in the At5g24530gene, LP primer (5′-caggtttatggcatatctcacgtc-3′) (SEQ ID NO: 108), incombination with the T-DNA right border primer, Tag3′(5′-tgataccagacgttgcccgcataa-3′) (SEQ ID NO: 109) or RB4(5′-tcacgggttggggtttctacaggac-3′) (SEQ ID NO: 110). The exact T-DNAinsertion in the second intron of At5g24530 was confirmed by sequencingof amplicons generated with the T-DNA primers from both the left andright border in combination with the gene specific primers LP or RP(5′-atgtccaagtccaatagccacaag-3′) (SEQ ID NO: 111).

cDNA Synthesis

RNA was isolated (from approximately 100 mg leaf tissue from 10 day oldseedlings) with the RNaesy kit (Qiagen, Venlo, The Netherlands) andtreated with the RNase-free DNase set (Qiagen). Total RNA was quantifiedusing an UVmini-1240 spectrophotometer (Shimadzu, Kyoto, Japan). cDNAwas synthesized with Superscript III reverse transcriptase (Invitrogen,Carlsbad, Calif., USA) and oligo(dT)15 (Promega, Madison, Wis., USA),according to the manufacturer's instructions.

Complementation of the Dmr6-1 Mutant

Complementation lines were generated by transforming dmr6 plants by thefloral dip method with Agrobacterium tumefaciens (Clough and Bent, 1998)containing the At5g24530 gene from Col-0 behind the 35S promoter. Theconstruct was generated by PCR amplification of the full lengthAt5g24530 from Col-0 cDNA with primers which included restriction sitesthat were used for directional cloning. A forward primer(5′-ttctgggatccaATGGCGGCAAAGCTGATATC-3′) (SEQ ID NO: 1) containing aBamHI restriction site near the start codon (ATG), amplified the 5′-endof DMR6 and at the 3′-end after the stop codon an EcoRI site wasgenerated with a reverse primer(5′-gatatatgaattcttagttgtttagaaaattctcgaggc-3′) (SEQ ID NO: 2). The35S-DMR6-Tn was cloned into the pGreenII0229 (Hellens, R. P., Edwards,E. A., Leyland, N. R., Bean, S., and Mullineaux, P. M. (2000)). pGreen:a versatile and flexible binary Ti vector for Agrobacterium-mediatedplant transformation. Plant Mol. Biol. 42, 819-832). 300 μMDL-Phosphinothricin (BASTA) resistant seedlings were isolated andanalyzed for H. parasitica susceptibility and for DMR6 expression levelsby RT-PCR.

Knock Down Lines of DMR6 by RNAi

RNAi lines were generated in the Ler eds 1-2 and Col-0 background. A 782bp long cDNA amplicon of Col-0 At5g24530 gene was generated. The PCR wasdone with the Phusion DNA polymerase (2 U/μL) and two different primercombinations. The amplicon from the first DMR6 gene specific primercombination

(RNAiDMR6F: (SEQ ID NO: 3) 5′-aaaaagcaggctGACCGTCCACGTCTCTCTGAA-3′ andRNAiDMR6R: (SEQ ID NO: 4) 5′-AGAAAGCTGGGTGAAACGATGCGACCGATAGTC-3′)was used as a template for the second PCR amplification with generalprimers allowing recombination into the pDONR7 vector of the GateWaycloning system. For the second PCR 10 μl of the first PCR (denaturationfor 30 sec. at 98° C. followed by 10 cycles of: 10 sec. at 98° C.; 30sec. at 58° C.; 30 sec. at 72° C.) in a total volume of 20 μl was usedas template. The second PCR (denaturation for 30 sec. at 98° C. followedby 5 cycles of: 10 sec. at 98° C.; 30 sec. at 45° C.; 30 sec. at 72° C.and 20 cycles of 10 sec. at 98° C.; 30 sec. at 55° C.; 30 sec. at 72° C.finished by a final extension of 10 min. at 72° C.) with the attB1(5′-GGGACAAGTTTGTACAAAAAAGCAGGCT-3′) (SEQ ID NO: 5) and the

attB2 (SEQ ID NO: 6) (5′-ggggaccactttgtacaagaaagctgggt-3′)were performed in a 50 μl reaction volume. PCR product was gel purifiedand 50 ηg insert was recombined into 150 ηg pDONR7 vector with theclonase BP enzyme. The vector was transformed into electrocompotent DH5aE. coli cells and plasmids containing the correct insert were isolatedand 100 ηg of the pDONR7 with the DMR6 amplicon were used in the LRreaction to recombine the insert in two opposite direction into 150 g3gpHellsgate8 vector. After transformation into E. coli, Spectomycinresistant clones were selected and the isolated plasmids were verifiedby a NotI digest for the right insert size and by colony PCR with asingle internal primer for At5G24530 (DfragmentF:5′-gagaagtgggatttaaaatagaggaa-3′) (SEQ ID NO: 7), if the inserts wasinserted twice in opposite direction an amplicon of 1420 bp could bedetected. Correct pHellsgate8 plasmids with the double insert inopposite directions were transformed into electrocompotent Agrobacteriumstrain, C58C1. Plasmids were isolated from the Agrobacterium andretransformed into the E. coli to confirm the right size of the plasmidand the insert by NotI digestion. The reconfirmed Agrobacterium strainswere used for the floral dip transformation of the Col-0 and Ler eds 1-2plants. The developed seeds were screened for Kanamycin resistance on½×GM plates, the Ti seedlings were transferred and the next generationof seeds the T2 was analysed for DMR6 expression and H. parasiticasusceptibility.

Gene Expression Profiling of the Dmr6 Mutant

Total RNA was isolated as described above. mRNA was amplified with theMessageAmp aRNA kit (Ambion). CATMA array (Crowe et al., 2003) slidescontaining approximately 25,000 gene specific tags were hybridizedaccording to standardized conditions described by de Jong et al. (deJong M., van Breukelen B., Wittink, F. R., Menke, F. L., Weisbeek, P.J., and Van den Ackerveken G. (2006). Membrane-associated transcripts inArabidopsis; their isolation and characterization by DNA microarrayanalysis and bioinformatics. Plant J. 46, 708-721). For quantitativePCR, cDNA templates were generated as described previously. Cyclethresholds were determined per transcript in triplicate using the ABIPRISM 7700 sequence detection system (Applied Biosystems, Foster City,Calif., USA) using SYBR Green I (Applied Biosystems, Foster City,Calif., USA) as reporter dye. Primer sets for the transcripts are DMR6(QDMR6F:5′-TGTCATCAACATAGGTGACCAG-3′ (SEQ ID NO: 8) and QDMR6R:5′-CGATAGTCACGGATTTTCTGTG-3′) (SEQ ID NO: 9), At1g14880(QAt1g14880F:5′-CTCAAGGAGAATGGTCCACA-3′ (SEQ ID NO: 10) and QAt1g14880R:5′-CGACTTGGCCAAATGTGATA-3′) (SEQ ID NO: 11), At4g14365 (QAt4g14365F:5′-TGGTTTTCTGAGGCATGTAAA-3′ (SEQ ID NO: 12) andQAt4g14365R:5′-AGTGCAGGAACATTGGTTGT-3′) (SEQ ID NO: 13), ACD6(QACD6F:5′-TGGACAGTTCTGGAGCAGAT-3′ (SEQ ID NO: 14) and QACD6R:5′-CAACTCCTCCGCTGTGAG-3′) (SEQ ID NO: 15), PR-5(QPR-5F:5′-GGCAAATATCTCCAGTATTCACA-3′ (SEQ ID NO: 16) and QPR-5R:5′-GGTAGGGCAATTGTTCCTTAGA-3′) (SEQ ID NO: 17), PR-2 (QPR-2F:5′-AAGGAGCTTAGCCTCACCAC-3′ (SEQ ID NO: 18) and QPR-2R:5′-GAGGGAAGCAAGAATGGAAC-3′) (SEQ ID NO: 19), PR-1(QPR-1F:5′-GAACACGTGCAATGGAGTTT-3′ (SEQ ID NO: 20) and QPR-1R:5′-GGTTCCACCATTGTTACACCT-3′) (SEQ ID NO: 21) and ACT-2 (QACT2F:5′-AATCACAGCACTTGCACCA-3′ (SEQ ID NO: 22) and QACT2R:5′-GAGGGAAGCAAGAATGGAAC-3′) (SEQ ID NO: 23) generating 100 base pairfragments.

Results Characterization of the Gene Responsible for Pathogen Resistancein the Dmr6 Mutant

Van Damme et al., 2005, supra disclose a dmr6 mutant that is resistantto H. parasitica. The level of resistance can be examined by countingthe number of sporangiophores per seedling seven day post inoculationwith the H. parasitica (isolate Waco9 or Cala2, obtainable from Dr. G.Van den Ackerveken, Plant-Microbe Interactions Group, University ofUtrecht, Utrecht, NL). The parental line, Ler eds 1-2 (Parker et al.,1996, Plant Cell 8:2033-2046), which is highly susceptible, is used as apositive control (and is set at 100%).

The reduction in sporangiophore formation on the infected dmr6 mutantscompared to seedlings of the parental lines is shown in FIG. 6A, whereinthe results of the quantification of Hyaloperonospora parasitica, Waco9sporulation (sporangiophores/seedling) on the downy mildew resistantdmr6-1 mutant, back-crossed twice to the parental line Ler eds 1-2, andon mutant dmr6-2 (FLAG_445D09 T-DNA line) compared to the control linesis shown.

According to the invention, the gene responsible for resistance to H.parasitica in the dmr6 mutants of van Damme et al., 2005, supra, hasbeen cloned by a combination of mapping and sequencing of candidategenes. Previously, the recessive dmr6 mutation was mapped near thenga139 marker on chromosome 5 to a region encompassing 74 genes. Finemapping linked the dmr6 locus to a mapping interval containing the BACsT13K7 and K18P6 between the markers At5g24420 and At5g24590 located inthe corresponding genes. This allowed the dmr6 interval to be confinedto a region of 18 candidate genes. Comparative sequence analysis of the18 genes in dmr6 and the parental line, Ler eds 1-2 revealed a pointmutation in the second exon of the At5g24530 gene. This single basechange of G to A, typical for an EMS mutation, changes a TGG a (trpcodon) to a TGA (premature stop codon) at nucleotide position 691 of thecoding sequence (FIG. 7). The early stop codon truncates the predictedoxidoreductase enzyme of 342 aa at position 141 before the conservedcatalytic domain suggesting that dmr6 is a null-allele. The At5g24530coding sequence (FIG. 2) is predicted to encode a protein with a mass of39.4 kDa. No biological role has so far been described for At5g24530.

At5g24530 is DMR6

A second allele, dmr6-2, was identified in a T-DNA insertion line(FLAG_445D09) from the mutant collection from INRA, Versailles. Thepresence and location of the T-DNA insert in the second intron ofAt5g24530 (FIG. 7) was confirmed by PCR and sequence analysis (data notshown). Progeny of the FLAG_445D09 line homozygous for the T-DNAinsertion was resistant to H. parasitica isolate Waco9, whereas theparental line (Ws-4) was susceptible (FIG. 6A). The At5g24530 transcriptcould be amplified by RT-PCR using primers in exon 2 and 3 in Ws-4, butnot in the homozygous dmr6-2 line (data not shown), indicating thatdmr6-2 can be considered a second null-allele.

To corroborate the idea that At5g24530 is required for susceptibility toH. parasitica the dmr6-1 mutant was transformed with the cDNA fromAt5g24530 cloned under control of the 35S promoter. In five independentdmr6-1 T2 seedlings the strong overexpression of At5g24530 was confirmedby RT-PCR (data not shown). All T3 lines, homozygous for the transgene,showed restoration of susceptibility to H. parasitica isolate Cala2(FIG. 6B), confirming that At5g24530 is DMR6. The complementation,together with the identification of two independent dmr6 mutants clearlyindicates that a functional DMR6 gene is required for susceptibility toH. parasitica.

DMR6 is Transcriptionally Activated During H. parasitica Infection

To study the expression of DMR6 during infection with H. parasiticarelative transcript levels were measured by quantitative PCR at sixdifferent time points from 0 days (2 hours) post inoculation to 5 dayspost inoculation (dpi) (FIG. 8). RNA was isolated from ten day old Lerseedlings that were spray inoculated with water (mock), compatible, orincompatible H. parasitica isolate. At 2 hours post inoculation (0 dpi)the levels of DMR6 transcripts were equal in the different treatments.Starting from 1 dpi, the level of DMR6 transcript was significantlyincreased in both the compatible and incompatible interaction comparedto mock-treated seedlings. The DMR6 transcript level was slightly butsignificantly higher at 1 dpi in the incompatible interaction (ACT of3.5, approximately 11 fold induction) than in the compatible (ACT of3.0, approximately 8 fold induction). The expression level increasedfurther in time to reach a stable high level at 4-5 dpi. At these timepoints the DMR6 transcript level was higher in the compatible than inthe incompatible interaction. The elevated DMR6 transcript levels duringcompatible and incompatible H. parasitica interactions suggest a role ofDMR6 in plant defence. The defence-associated expression of DMR6 couldbe confirmed in our three enhanced-defence mutants, dmr3, dmr4, and dmr5(Van den Ackerveken et al., unpublished). Furthermore, in silicoanalysis of DMR6 levels in the Genevestigator Mutant Surveyor(Zimmermann,P., Hennig, L., and Gruissem, W. (2005). Gene-expressionanalysis and network discovery using Genevestigator. Trends Plant Sci.10, 407-409) showed that the gene is strongly induced in the pathogenresistant mutants mpk4 and cpr5. In the cpr5/npr1 double mutant the DMR6transcript level remained high indicating that the induction of DMR6expression is mostly NPR1 independent. Salicylic acid appears to be animportant signal in the induction of DMR6 expression during senescenceas nahG transgenic plants (expressing the bacterial salicylatehydroxylase gene) showed only low levels of DMR6 transcript.

To investigate in more detail how the expression of DMR6 is activatedduring biotic and abiotic stress, DMR6 reporter lines were generated.The localisation of DMR6 expression was studied in transgenic Col-0 andLer eds 1-2 plants containing the DMR6 promoter linked to the uidA(β-glucuronidase, GUS) reporter gene (pDMR6::GUS). To visualise both H.parasitica hyphal growth, by staining with trypan blue, as well as GUSactivity, magenta-Xgluc was used as a 0-glucuronidase substrate yieldinga magenta precipitate. In uninfected plants no GUS expression could bedetected in the different plant organelles; roots, meristem, flower,pollen and seed. The expression of DMR6 was induced in the compatibleinteractions, Ler eds 1-2 infected with Cala2 (FIG. 9A), and Col-0infected with isolate Waco9 (FIG. 9B). GUS expression was also inducedin the incompatible interaction Ler eds 1-2 inoculated with isolateEmoy2 (FIG. 9C). As shown in FIGS. 9A and 9B DMR6 expression wasconfined to the cells in which H. parasitica had formed haustoria. Plantcells containing the most recently formed haustoria did not showdetectable levels of GUS activity (FIG. 9A, indicated by asterisk).During the incompatible interaction (FIG. 9C) activity of the DMR6promoter could only be detected in the cells that were in contact withthe initial invading hyphae. In death cells, resulting from thehypersensitive response (HR, visualized by trypan blue stainingindicated in FIG. 9C by asterisk) no GUS activity could be detected,possibly due to protein degradation in these cells. To test if the DMR6expression in haustoria-containing cells is caused by a wound-likeresponse, seedlings were wound by incision with scissors and stained forGUS activity 3 days later. No detectable promoter DMR6 GUS expressionwas seen, indicating that the expression of DMR6 is not induced bywounding (FIG. 9D). Furthermore the local induction of DMR6 expressionwas tested in response to treatment with benzothiadiazole (BTH), afunctional analogue of salicylic acid (SA). At 3 days post BTH treatmentGUS activity was mainly localized in the newly formed, but not in themature leaves (FIG. 9E). Analysis of pDMR6::GUS lines confirm theexpression data described above and highlights the strictly localizedinduction of DMR6 in response to H. parasitica infection.

The Dmr6-1 Mutant Constitutively Expresses Defence AssociatedTranscripts

To elucidate how the lack of DMR6 results in H. parasitica resistance,the transcriptome of the dmr6-1 mutant compared to the Ler eds 1-2parental line was analysed. Probes derived from mRNA of the above-groundparts of 14 day old dmr6-1 and Ler eds 1-2 seedlings were hybridised onwhole genome CATMA micro arrays. A total of 58 genes were found to besignificantly differentially expressed in dmr6-1, of which 51 genes hadelevated and 7 genes had reduced transcript levels. A pronounced set ofthe 51 induced transcripts have been identified as genes associated withactivated plant defence responses, e.g., ACD6, PR-5, PR-4/HEL and PAD4.These data indicate that the loss of DMR6 results in the activation of aspecific set of defence-associated transcripts. The finding that DMR6 isamong the dmr6-1-induced genes corroborates the idea that DMR6 isdefence-associated. To test if the induced expression of thedefence-associated genes was due to the loss of DMR6 and not due toadditional ethane methyl sulfonate (EMS) mutations remaining in thebackcrossed dmr6-1 mutant the transcript level of a selection of genes(At4g14365, At1g14880, ACD6, PR-1, PR-2 and PR-5) was verified byquantitative PCR in both the dmr6-1 and dmr6-2 mutant (FIGS. 10A and10B). We could only test DMR6 transcript levels in the dmr6-1 mutant(FIG. 10A) as the dmr6-2 mutant (FIG. 10B) has a T_DNA insertiondisrupting the DMR6 transcript. The induction of DMR6 as observed in themicro array analysis was confirmed by Q-PCR in dmr6-1 compared to Lereds 1-2 (FIG. 10A). FIGS. 10A and 10B show that all six selected geneswere elevated in both dmr6 mutants compared to the parental lines. Theobserved elevated expression of the selected defence-associated genes inthe dmr6 mutants indicates that lack of DMR6 activates a plant defenceresponse. The activation of this set of defence-associated transcriptscould be the primary cause of resistance to H. parasitica in the dmr6mutants.

Example 2 Identification of DMR6 Orthologs in Crops 1. Screening ofLibraries on the Basis of Sequence Homology

The nucleotide and amino acid sequences of the DMR6 coding sequence andprotein of Arabidopsis thaliana are shown in FIG. 2. Public libraries ofnucleotide and amino acid sequences were compared with the sequences ofFIG. 2. This comparison resulted in identification of the complete DMR6coding sequences and predicted amino acid sequences in Aquilegiaspecies, Citrus sinensis, Coffea canephora, Cucumis sativus, Gossypiumhirsutum, Lactuca sativa, Medicago truncatula, Oryza sativa (3), Populustrichocarpa (2), Solanum lycopersicum (2), Sorghum bicolor, Spinaciaoleracea, Vitis vinifera, Zea mays, and Zingiber officinale. Thesequence information of the orthologous proteins thus identified isgiven in Table 1 and visualized in a multiple alignment in FIG. 1. Formany other plant species, orthologous DNA fragments could be identifiedby BlastX as reciprocal best hits to the Arabidopsis or other plant DMR6protein sequences.

Table 1 lists the GI numbers (GenInfo identifier) and Genbank accessionnumber for Expressed Sequence Tags (ESTs) and mRNA or protein sequencesof the Arabidopsis DMR6 mRNA and orthologous sequences from other plantspecies. A GI number (GenInfo identifier, sometimes written in lowercase, “gi”) is a unique integer which identifies a particular sequence.The GI number is a series of digits that are assigned consecutively toeach sequence record processed by NCBI. The GI number will thus changeevery time the sequence changes. The NCBI assigns GI numbers to allsequences processed into Entrez, including nucleotide sequences fromDDBJ/EMBL/GenBank, protein sequences from SWISS-PROT, PIR and manyothers. The GI number thus provides a unique sequence identifier whichis independent of the database source that specifies an exact sequence.If a sequence in GenBank is modified, even by a single base pair, a newGI number is assigned to the updated sequence. The accession numberstays the same. The GI number is always stable and retrievable. Thus,the reference to GI numbers in the table provides a clear andunambiguous identification of the corresponding sequence.

TABLE 1 Genbank accession numbers and GenInfo identifiers of theArabidopsis Arabidopsis DMR6 mRNA and orthologous sequences from otherplant species Species Common name Detail GI number Genbank Arabidopsisthaliana Thale cress mRNA 42568064 NM_122361 Aquilegia_sp Aquilegia ESTs75461114 DT768847.1 74538666 DT745001.1 74562677 DT760187.1 75461112DT768846.1 74562675 DT760186.1 Citrus sinensis Sweet Orange ESTs 5793134CX672037.1 57933368 CX673829.1 63078039 CX309185.1 Coffea canephoraCoffea ESTs 82485203 DV705375.1 82458236 DV684837.1 82461999 DV688600.182487627 DV707799.1 Gossypium hirsutum Cotton ESTs 109842586 DW241146.148751103 CO081622.1 Sorghum bicolor Sorghum ESTs 45992638 CN150358.157813436 CX614669.1 45985339 CN145819.1 57821006 CX622219.1 45989371CN148311.1 57821495 CX622708.1 45959033 CN130459.1 45985193 CN145752.118058986 BM322209.1 45958822 CN130381.1 30164583 CB928312.1 Medicagotruncatula Barrel medic Genome MtrDRAFT_AC119415g1v1 draft protein92878635 ABE85154 Oryza sativa 1 Rice Genome OSJNBb0060I05.4 protein18057095 AAL58118.1 Oryza sativa 2 mRNA 115450396 NM_001055334 protein115450397 NP_001048799 Oryza sativa 3 mRNA 115460101 NM_001060186protein 115460102 NP_001053651 Populus trichocarpa 1 Poplar Genome:LG_XII: 3095392-3103694 protein: Poptr1_1: 569679, eugene3.00120332Populus trichocarpa 2 Poplar Genome: LG_XV: 201426-209590 protein:Poptr1_1: 732726, estExt_Genewise1_v1.C_LG_XV0083 Solarium lycopersicum1 Tomato ESTs 62932307 BW689896.1 58229384 BP885913.1 117682646DB678879.1 5894550 AW035794.1 117708809 DB703617.1 62934028 BW691617.115197716 BI422913.1 4381742 AI486371.1 5601946 AI896044.1 4387964AI484040.1 4383017 AI487646. 5278230 AI780189.1 12633558 BG133370.176572794 DV105461.1 117692514 DB718569.1 4385331 AI489960.1 4383253AI487882.1 4384827 AI489456.1 Solarium lycopersicum 2 Tomato ESTs47104686 BT013271.1 14685038 BI207314.1 14684816 BI207092.1 Zea maysMaize ESTs 110215403 EC897301.1 76291496 DV031064.1 91050479 EB160897.191874282 EB404239.1 110540753 EE044673.1 78111856 DV530253.1 94477588EB706546.1 71441483 DR822533.1 78111699 DV530096.1 78107139 DV525557.176017449 DT944619.1 91048249 EB158667.1 78104908 DV523326.1 78088214DV516607.1 76291495 DV031063.1 71441482 DR822532.1 78088213 DV516606.1Vitis vinifera Grape ESTs 33396402 CF202029.1 33399765 CF205392.145770972 CN006824.1 45770784 CN006636.1 45770528 CN006380.1 45770631CN006483.1 33400623 CF206250.1 33396335 CF201962.1 30134763 CB920101.130305300 CB982094.1 71857419 DT006474.1 30305235 CB982029.1 Zingiberofficinale Ginger ESTs 87108948 DY375732.1 87095447 DY362231.1 87095448DY362232.1 87094804 DY361588.1 87095449 DY362233.1 87094803 DY361587.1Lactuca sativa Lettuce Sequence described in this patent applicationSpinacia oleracea Spinach Sequence described in this patent applicationCucumis sativus Cucumber Sequence described in this patent applicationNicotiana benthamiana Tobacco Sequence described in this patentapplication

Identification of Orthologs by Means of Heterologous Hybridisation

The DMR6 DNA sequence of Arabidopsis thaliana as shown in FIG. 2 is usedas a probe to search for homologous sequences by hybridization to DNA ofany plant species using standard molecular biological methods. Usingthis method orthologous genes are detected by southern hybridization onrestriction enzyme-digested DNA or by hybridization to genomic or cDNAlibraries. These techniques are well known to the person skilled in theart. As an alternative probe the DMR6 DNA sequence of any other moreclosely related plant species can be used as a probe.

3. Identification of Orthologs by Means of PCR

For many crop species, partial DMR6 mRNA or gene sequences are availablethat are used to design primers to subsequently PCR amplify the completecDNA or genomic sequence. When 5′ and 3′ sequences are available themissing internal sequence is PCR amplified by a DMR6 specific 5′ forwardprimer and 3′ reverse primer. In cases where only 5′, internal or 3′sequences are available, both forward and reverse primers are designed.In combination with available plasmid polylinker primers, inserts areamplified from genomic and cDNA libraries of the plant species ofinterest. In a similar way, missing 5′ or 3′ sequences are amplified byadvanced PCR techniques; 5′RACE, 3′ RACE, TAIL-PCR, RLM-RACE orvectorette PCR.

As an example the sequencing of the Lactuca sativa (lettuce) DMR6 cDNAis provided. From the Genbank EST database at NCBI several Lactuca DMR6ESTs were identified using the tblastn tool starting with theArabidopsis DMR6 amino acid sequence. Clustering and alignment of theESTs resulted in a consensus sequence for a 5′ DMR6 fragment. To obtainthe complete lettuce DMR6 cDNA the RLM-RACE kit (Ambion) was used onmRNA from lettuce seedlings. The 3′ mRNA sequence was obtained by usingtwo primers that were designed in the 5′ DMR6 consensus sequence derivedfrom ESTs (Lsat_dmr6_fw1: CGATCAAGGTCAACACATGG (SEQ ID NO: 24), andLsat_dmr6_fw2: TCAACCATTACCCAGTGTGC) (SEQ ID NO: 25) and the 3′RACEprimers from the kit. Based on the assembled sequence new primers weredesigned to amplify the complete DMR6 coding sequence from cDNA toprovide the nucleotide sequence and derived protein sequence aspresented in FIG. 3.

The complete DMR6 coding sequences from more than 10 different plantsspecies have been identified from genomic and EST databases. From thealignment of the DNA sequences, conserved regions in the coding sequencewere selected for the design of degenerate oligonucleotide primers (forthe degenerate nucleotides the abbreviations are according to the IUBnucleotide symbols that are standard codes used by all companiessynthesizing oligonucleotides; G=Guanine, A=Adenine, T=Thymine,C=Cytosine, R=Aor G, Y=C orT, M=Aor C, K=G or T, S=C orG, W=Aor T, B=Cor G or T, D=G or A or T, H=A or C or T, V=Aor C or G, N=A or C or G orT).

The procedure for obtaining internal DMR6 cDNA sequences of a givenplant species is as follows:

1. mRNA is isolated using standard methods,2. cDNA is synthesized using an oligo dT primer and standard methods,3. using degenerate forward and reverse oligonucleotides a PCR reactionis carried out,4. PCR fragments are separated by standard agarose gel electrophoresisand fragments of the expected size are isolated from the gel,5. isolated PCR fragments are cloned in a plasmid vector using standardmethods,6. plasmids with correct insert sizes, as determined by PCR, areanalyzed by DNA sequencing,7. Sequence analysis using blastX reveals which fragments contain thecorrect internal DMR6 sequences,8. The internal DNA sequence can then be used to design gene- andspecies-specific primers for 5′ and 3′ RACE to obtain the complete DMR6coding sequence by RLM-RACE (as described above).

As an example the sequencing of the Cucumis sativus (cucumber) DMR6 cDNAis provided. For cucumber several primer combinations between thefollowing primers were successful in amplifying a stretch of internalcoding sequence from cDNA; forward primers dmr6_deg_fw1B(TTCCAGGTDATTAAYCAYGG) (SEQ ID NO: 26), dmr6_deg_fw2BCATAAYTGGAGRGAYTAYCT) (SEQ ID NO: 27), dmr6_deg_fw3B(GARCAAGGRCARCAYATGGC) (SEQ ID NO: 28) and dmr6_deg_fw4(AATCCTCCTTCHTTCAAGGA) (SEQ ID NO: 29) and reverse primers dmr6_deg_rv3B(AGTGCATTKGGGTCHGTRTG) (SEQ ID NO: 30), dmr6_deg_rv4(AATGTTRATGACAAARGCAT) (SEQ ID NO: 31) and dmr6_deg_rv5(GCCATRTGYTGYCCTTGYTC) (SEQ ID NO: 32). After cloning and sequencing ofthe amplified fragments cucumber DMR6-specific primers were designed for5′ RACE (Cuc_dmr6_rv1: TCCGGACATTGAAACTTGTG (SEQ ID NO: 33) and Cuc_dmr6rv2: TCAAAGAACTGCTTGCCAAC) (SEQ ID NO: 34) and 3′ RACE (Cuc_dmr6_fw1:CGCACTCACCATTCTCCTTC (SEQ ID NO: 35) and Cuc_dmr6_fw2:GGCCTCCAAGTCCTCAAAG) (SEQ ID NO: 36). Finally the complete cucumber DMR6cDNA sequence was amplified and sequenced (FIG. 5). A similar approachwas a used for spinach, Spinacia oleracea (FIG. 4), Solanum lycopersicum(FIG. 12) and Nicotiana benthamiana (FIG. 13).

Orthologs identified as described in this example can be modified usingwell-known techniques to induce mutations that reduce the DMR6expression or activity, to obtain non-genetically modified plantsresistant to Fungi or Oomycota. Alternatively, the genetic informationof the orthologs can be used to design vehicles for gene silencing, andto transform the corresponding crop plants to obtain plants that areresistant to Oomycota.

Example 3 Mutation of Seeds

Seeds of the plant species of interest are treated with a mutagen inorder to introduce random point mutations in the genome. Mutated plantsare grown to produce seeds and the next generation is screened for theabsence of reduction of DMR6 transcript levels or activity. This isachieved by monitoring the level of DMR6 gene expression, or bysearching for nucleotide changes (mutations) by the TILLING method, byDNA sequencing, or by any other method to identify nucleotide changes.The selected plants are homozygous or are made homozygous by selfing orinter-crossing. The selected homozygous plants with absent or reducedDMR6 transcript activity are tested for increased resistance to thepathogen of interest to confirm the increased disease resistance.

Example 4

Transfer of a Mutated Allele into the Background of a Desired Crop

Introgression of the desired mutant allele into a crop is achieved bycrossing and genotypic screening of the mutant allele. This is astandard procedure in current-day marker assistant breeding of crops.

Example 5 Use of the DMR6 Promoter for Pathogen-Induced Gene Expressionand the Generation of Disease Resistant Plants

Precise control of transgene expression is pivotal to the engineering ofplants with increased disease resistance. In the past, constitutiveoverexpression of transgenes frequently has resulted in poor qualityplants. It has therefor been suggested to use pathogen-induciblepromoters, by which the transgenes are expressed only when and wherethey are needed—at infection sites.

Local and inducible expression of engineered genes, e.g. master switchgenes, elicitor or Avr genes, anti-microbial genes, or toxic genes,results in the activation of defence or cell death that will lead topathogen resistance, such as described by Gurr and Rushton (Trends inBiotechnology 23: 275-282, 2005). A good example is provided by De wit(Annu. Rev. Phytopathol. 30: 391-418, 1992) who proposes the use of theAvr9-Cf9 combination to achieve induced cell death leading to diseaseresistance. The tissue-specificity and inducibility of expression is ofprime importance for such approaches, as described by Gurr and Rushton(Trends in Biotechnology 23: 283-290, 2005).

According to the present invention, the DMR6 promoter has beendemonstrated to show a strong, inducible, localized expression based onpromoter-GUS analysis. Thus, the DMR6 promoter is very suitable forengineering disease resistance in transgenic plants. The DMR6 promoterconsists of a region of 2.5 kb that is upstream of the Arabidopsis DMR6coding sequence (ATG start codon) and includes the 5′UTR (as depicted inFIG. 11). This pathogen-inducible promoter is then used to engineersuitable transgene constructs, using standard techniques known theperson skilled in the art.

Using orthologous DNA sequences from a given plant species primers aredesigned for PCR. These are then used to screen genomic libraries of theplant species of interest to identify the genomic clones that containthe DMR6 ortholog with its promoter and regulatory sequences.Alternatively, the genomic clones are isolated by screening a librarywith a labelled PCR fragment corresponding to the DMR6 orthologous gene.Sequencing reveals the nucleotide sequence of the promoter. The regionof 2-5 kb upstream the DMR6 orthologous coding sequence (ATG startcodon), so including the 5′UTR, is then amplified by PCR to engineertransgene constructs for plant transformation.

Example 6

This example demonstrates the complementation of mutant dmr6-1 inArabidopsis thaliana by DMR6 orthologs from 4 different crop species.For this, DMR6 orthologs of Cucumis sativa (Cs), Spinacia oleracea (So),Lactuca sativa (Ls) and Solanum lycopersicum (Sl) were cloned into aplant expression vector under the control of the 35S promoter and,subsequently, this vector was transformed into a Arabidopsis thalianamutant dmr6-1.

Briefly, mRNA was isolated using standard methods and cDNA wassynthesized using an oligo dT primer and standard methods. Subsequently,PCR fragments were generated using primer pairs for each crop asdepicted in Table 3 below. The generated PCR products were cloned into apENTR/D-TOPO vector using the pENTR/D-TOPO cloning kit from Invitrogenand resulting plasmids with correct insert sizes, as determined by PCR,were analyzed by DNA sequencing. Recombination to the pB7WG2,0 vectorwas done using LR clonase II from Invitrogen and the resulting plasmidswere analyzed by PCR and digestion with restriction enzymes. Suitableplasmids were transformed into Agrobacterium tumefaciens C58C1 PGV2260and plasmids from Agrobacterium were analyzed by PCR and digestion withrestriction enzymes.

TABLE 3 Primer pairs for cloning dmr6 orthologs in a suitable plantexpression vector. Arabidopsis AtDMR6_fw CACCATGGCGGCAAAGCTGAT thalianaA (SEQ ID NO: 85) AtDMR6UTR_rv GACAAACACAAAGGCCAAAGA (SEQ ID NO: 86)Cucumis sativa cuc_fw CACCATGAGCAGTGTGATGGA GAT (SEQ ID NO: 87)cucUTR_rv TGGGCCAAAAAGTTTATCCA (SEQ ID NO: 88) Spinacia oleracea spi_fwCACCATGGCAAACAAGATATT ATCCAC (SEQ ID NO: 89) spiUTR_rvTTGCTGCCTACAAAAGTACAA A (SEQ ID NO: 90) Lactuca sativa Lsat_fwCACCATGGCCGCAAAAGTCAT CTC (SEQ ID NO: 91) LsatUTR_rvCATGGAAACACATATTCCTTCA (SEQ ID NO: 92) Solanum Slyc1dmr6_fwCACCATGGAAACCAAAGTTAT lycopersicum TTCTAGC (SEQ ID NO: 93)Slyc1dmr6UTR_rv GGGACATCCCTATGAACCAA (SEQ ID NO: 94)

Arabidopsis thaliana dmr6-1 plants were transformed with the aboveconstructs by dipping into Agrobacterium solution and overexpression ofcrops DMR6 in Arabidopsis T1 plants is verified by RT-PCR using thecrops DMR6 cloning primers (Table 3). Finally, Arabidopsis T2 and T3plants were infected with Hyaloperonospora parasitica Cala2 to confirmcomplementation. The results are shown in FIG. 14.

As shown in FIG. 14, all DMR6 orthologs tested were capable ofcomplementing Arabidopsis thaliana mutant dmr6-1 indicating that theDMR6 orthologs identified encode DMR6 proteins with a similarfunctionality as Arabidopsis thaliana DMR6.

Example 7

Mutations in the Brassica oleracea DMR6 Genes, DMR6-1 and DMR6-2, Resultin Downy Mildew Resistance

Experimental Procedures Mutation of Seeds

A rapid cycling Brassica oleracea line that is fully susceptible todowny mildew (i.e., Hyaloperonospora parasitica/Hyaloperonosporabrassicae) (FastPlantsR, https://fastplants.org/origin/) was used togenerate DMR6 mutant lines. B. oleracea contains two DMR6 genes,designated DMR6-1 (BoDMR6_Scf108) and DMR6-2 (BoDMR6_Scf155). The rapidcycling B. oleracea is used as a model plant and as a research tool forB. oleracea, since it is representative of all B. oleracea species. Therapid cycling line is able to interbreed with each other B. oleraceaspecies. It is well known in the art that results obtained using therapid cycling B. oleracea line are predictive for all B. oleraceaspecies. Knock out mutants with premature stop codons were generated inboth genes using EMS mutagenesis, and the mutants in DMR6-1 weredesignated “bodmr6-1” while the mutants in DMR6-2 were designated“bodmr6-2”. Table 4 shows the sequence details for the mutations in theDMR6-1 and DMR6-2 genes of B. oleracea, whereby three differentmutations were identified in the DMR6-1 gene and only one mutation wasidentified in DMR6-2.

TABLE 4 Mutations in the DMR6-1 and DMR6-2 genes of the Brassicaoleracea rapid cycling line CDS Nucleotide Amino Acid SNP Name in AllelePosition Change Change Protein bodmr6-1-1 181 C to T R to STOP R61*bodmr6-1-2 691 C to T Q to STOP Q234* bodmr6-1-3 714 G to A W to STOPW201* bodmr6-2 368 G to A W to STOP W123*

Briefly, the bodmr6-1-1 mutation was at position 181 in the DMR6-1 gene,and it turned a Cytosine (C) into a Thymine (C to T mutation). Thismutation resulted in a change at the amino acid level, converting anArginine (R) at position 61 in the protein into a premature stop codon(R61*). The bodmr6-1-2 mutation was a C to T mutation at position 691 inthe DMR6-1 gene that resulted in a change at the amino acid level from aGlutamine (Q) at position 234 in the protein to a premature stop codon(Q234*). The bodmr6-1-3 mutation was a Guanine (G) to an Adenine (G to Amutation) at position 714 in the DMR6-1 gene that resulted in a changeat the amino acid level from a Tryptophan (W) at position 201 in theprotein to a premature stop codon (W201*). Finally, the bodmr6-2mutation was a G to A mutation at position 368 in the DMR6-2 gene, whichresulted in a change at the amino acid level from a W at position 123 inthe protein to a premature stop codon (W123*). These identifiedmutations are located before an essential iron binding residue which isconserved in all 20G oxygenases; without this residue, 20G oxygenasessuch as DMR6 are catalytically inactive (Wilmouth et al. (2002),Structure, 10:93-103). Therefore, these premature stop codons cause acomplete loss of enzyme function.

FIGS. 22A-22F show the nucleotide alignment of the DMR6-1 mutant alleleswith the corresponding wild type sequence of DMR6-1, whereby the mutantalleles (BoDMR6_C181T_scf=SEQ ID NO: 128; BoDMR6_C691T_scf=SEQ ID NO:129; BoDMR6_G714A_scf=SEQ ID NO: 130) are shown below the wild typesequence (BoDMR6_WT_scf108=SEQ ID NO: 114). FIGS. 23A-23E show thenucleotide alignment of the DMR6-2 mutant allele with the correspondingwild type sequence of DMR6-2, whereby the mutant allele(BoDMR6_G368A_scf=SEQ ID NO: 131) is shown below the wild type sequence(BoDMR6_WT_scf155=SEQ ID NO: 115). FIGS. 24A-24B show the polypeptidealignment of the DMR6-1 mutant polypeptides with the corresponding wildtype polypeptide of DMR6-1, whereby the mutant polypeptides(BoDMR6_C181T_scf=SEQ ID NO: 141; BoDMR6_C691T_scf=SEQ ID NO: 142;BoDMR6_G714A_scf=SEQ ID NO: 143) are shown below the wild typepolypeptide (BoDMR6_WT_scf108=SEQ ID NO: 112). FIGS. 25A-25B show thepolypeptide alignment of the DMR6-2 mutant polypeptide with thecorresponding wild type polypeptide of DMR6-2, whereby the mutantpolypeptide (BoDMR6_G368A_scf=SEQ ID NO: 144) is shown below the wildtype polypeptide (BoDMR6_WT_scf155=SEQ ID NO: 113).

Creating Homozygous DMR6 Mutant Plants

Plants carrying the bodmr6-1-1 and bodmr6-2 mutant alleles were crossedto produce a first segregating F2 population (F2 pop. 1). This F2population was genotyped to identify the presence of the mutant DMR6-1and mutant DMR6-2 alleles. Two different genotypes were distinguished,as follows: wild type (i.e., DMR6-1R6-DMR6-1; DMR6-2/DMR6-2), referredto as “AA BB”, and homozygous double mutant (bodmr6-1-1/bodmr6-1-1;bodmr6-2/bodmr6-2). Plants with these two genotypes were then used intest assays.

Plants carrying the bodmr6-1-2 allele were crossed with plants carryingthe bodmr6-2 mutant allele to produce a second segregating F2 population(F2 pop. 2). This F2 population was genotyped to identify the presenceof the mutant DMR6-1 and mutant DMR6-2 alleles. Two different genotypeswere distinguished, as follows: wild type (i.e., DMR6-1R6-1/DMR6-1;DMR6-2/DMR6-2), referred to as “AA BB”, and homozygous double mutant(bodmr6-1-2 bodmr6-1-2; bodmr6-2 bodmr6-2). Plants with these twogenotypes were then used in test assays.

Plants carrying the bodmr6-1-3 allele were crossed with plants carryingthe bodmr6-2 mutant allele to produce a third segregating F2 population(F2 pop. 3). This F2 population was genotyped to identify the presenceof the mutant DMR6-1 and mutant DMR6-2 alleles. Two different genotypeswere distinguished, as follows: wild type (i.e., DMR6-1/DMR6-1;DMR6-2/DMR6-2), referred to as “AA BB”, and homozygous double mutant(bodmr6-1-3 bodmr6-1-3; bodmr6-2 bodmr6-2). Plants with these twogenotypes were then used in test assays.

Genotyping

Plants were genotyped by PCR and High-Resolution Melting Curve (HRM)using primers and probes (listed in Table 5A and Table 5B) designed toidentify the mutant alleles listed in Table 4.

TABLE 5A Primers used for genotyping Allele Forward Reverse bodmr6-AGTCCAACAAATCCACCAAGC CCTTTTCAGGCACATAAGTTCA 1-1 (SEQ ID NO: 116)(SEQ ID NO: 117) bodmr6- ATTCTTCTCCAAGATGCTACCG GGATTAACGGCGAACCACT 1-2(SEQ ID NO: 118) (SEQ ID NO: 119) bodmr6- CCAAGATGCTACCGTTTGTGTTGATGACAAAAGCATCAGGA 1-3 (SEQ ID NO: 120) (SEQ ID NO: 121) bodmr6-CCGAGATTATCGACGAGCTT TTGTCGATAGGATAACAATGA 2 (SEQ ID NO: 122)AGTC (SEQ ID NO: 123)

TABLE 5B Probes used for genotyping Allele Probe bodmr6-1-1GCTTGCTCCTGATTCGGGC (SEQ ID NO: 124) bodmr6-1-2CGTTTGTGGTCTATAGATCTTGATCGT (SEQ ID NO: 125) bodmr6-1-3CAGTGATTCGCCGTTAATCCACC (SEQ ID NO: 126) bodmr6-2GAAGAGGTTAATAATTAGAGAGACTATCTC (SEQ ID NO: 127)

The full sequence of the dmr6-1-1 mutant allele is SEQ ID NO: 128, thefull sequence of the dmr6-1-2 mutant allele is SEQ ID NO: 129, the fullsequence of the dmr6-1-3 mutant allele is SEQ ID NO: 130, and the fullsequence of the dmr6-2 mutant allele is SEQ ID NO: 131.

Whole Plant Downy Mildew Test

The F2 pop. 1 B. oleracea plants described above were used to performthe whole plant downy mildew test, which enables phenotyping withoutwounding or damaging plant tissues. Plants with the first leaf pair wereused, as this is the optimal developmental stage for a downy mildewtest. Thirteen homozygous mutant plants and fifteen wild type plantswere tested.

Plants were inoculated with downy mildew isolate Hyaloperonosporaparasitica/Hyaloperonospora brassicae Y113 (collected on B. oleracea inDannstadt, Germany). At time point 0, plants were inoculated with 2×10⁴spores (20,000 spores/mL). Once infected, plants were kept in a plastictent built in a climate cell, which was kept at 15° C. with a day-nightcycle of 16 hours light-8 hours dark (16:8 cycle). The plants werescored 8 to 9 days after inoculation using the scoring scale shown inTable 6, below.

TABLE 6 Whole plant downy mildew test and leaf disc test scoring scaleScore Phenotype leaves/leaf discs Assessment 1 100% sporulation, coveredwith spores Susceptible on both side of the leaf tissue 3 50%sporulation on both side of the leaf Susceptible tissue 5 Less than 25%sporulation on one side Susceptible of the leaf tissue 7 No sporulation,some necrosis Resistant 9 No sporulation/no necrosis Resistant *Evendisease scores of 2, 4, 6, and 8 are used to designate intermediatedisease severity, i.e., a severity that is in between the neighbouringodd scores.

Leaf Disc Downy Mildew Test

The F2 pop. 1, F2 pop. 2, and F2 pop. 3 B. oleracea plants describedabove were used in the leaf disc assay, which allows high-throughputdisease testing. Plants with four true leaves were used, as this is theoptimal developmental stage for leaf disc sampling.

Plants were inoculated with downy mildew isolate Hyaloperonosporaparasitica/Hyaloperonospora brassicae Y113 (collected on B. oleracea inDannstadt, Germany). At time point 0, plants were inoculated with 2×10⁴spores (20,000 spores/mL). Once infected, plants were kept in a plastictent built in a climate cell, which was kept at 15° C. with a day-nightcycle of 16 hours light-8 hours dark (16:8 cycle). The plants werescored 8 to 9 days after inoculation using the scoring scale shown inTable 6.

Results

FIG. 15 shows the results of the whole plant downy mildew test on thesegregating F2 population 1. The average score of wild type (“AA BB”)plants are clearly distinct from the average scores of homozygous doublemutant plants (“aa bb”). Specifically, wild type plants have an averagescore of 4 (i.e., susceptible), while homozygous double mutant plantshave an average score of about 8 (i.e., resistant). These resultsdemonstrate that homozygous double mutants were resistant to downymildew. The phenotypes of exemplary wild type (“AA BB”) and homozygousdouble mutant (“aa bb”) leaves inoculated with downy mildew are shown inFIG. 16.

FIGS. 17A-17C show the results of the leaf disc downy mildew test on thesegregating F2 pop. 1, F2 pop. 2, and F2 pop. 3. In all threesegregating F2 populations, the wild type plants (“AA BB”) weresusceptible (score of about 1), and the double mutant plants (“aa bb”)were resistant (score of about 6 to 7). All three DMR6-1 mutant alleles(i.e., bodmr6-1-1, bodmr6-1-2, and bodmr6-1-3), conferred resistancewhen a DMR6-2 mutant allele (i.e., bodmr6-2) was also present.

These results show that homozygous double mutants with all threecombinations of mutant alleles (bodmr6-1-1 and bodmr6-2; bodmr6-1-2 andbodmr6-2; or bodmr6-1-3 and bodmr6-2) in B. oleracea are resistant todowny mildew when measured using the leaf disc test. In addition,homozygous double mutants with the alleles bodmr6-1-1 and bodmr6-2 in B.oleracea are resistant to downy mildew when measured using the wholeplant test.

Example 8

Brassica oleracea Plants with a Double Knockout of DMR6-1 and DMR6-2have Intermediate Resistance to Xanthomonas campestris Campestris andResistance to Downy Mildew

Background

The bacterial pathogen Xanthomonas campestris campestris (Xcc) is amajor problem in areas with warm and wet conditions. Xcc infects plantleaves by wounds or through the hydathodes, and Xcc race 1 (Xcc 1) andrace 4 (Xcc 4) are the most prevalent Xcc races in areas where Brassicacrops are grown. Xcc race determination is based on Vicente et al.(2013), Molecular Plant Pathology, 14(1): 2-18.

Experimental Procedures Plasmids for Gene Editing

Plasmids obtained and methods used were essentially as described byBelhaj et al. (2013), Plant Methods, 9:39. A plasmid with a customizedsingle guide RNA (sgRNA) was created using an oligo (SEQ ID NO: 132).The newly created plasmid and plasmid pICSL11021 (containing CAS9;https://www.addgene.org/49771/) were harvested from E. coli culturesusing the Qiagen Maxi prep kit. Plasmids were checked via both Sangersequencing and restriction analysis and subsequently used fortransfection. Mutations obtained in the DMR6-1 and DMR6-2 genes via geneediting (GE) all resulted in frameshift mutations leading to prematurestop codons.

Protoplast Transfection and Regeneration

The starting material used for protoplast isolation was 16 to 19 day oldleaves of cauliflower (i.e., Brassica oleracea) double haploid (DH) line194190 in vitro seedlings (susceptible to downy mildew and Xcc). Theprotoplast isolation protocol was as follows:

-   -   1. Cut leaves feather-like in TVL, and keep in dark for 1 hour.    -   2. Replace TVL with 15 ml 0.6% enzyme solution, and incubate        overnight in the dark at 25° C.    -   3. Filter liquids and tissue residue through 100 μm filter        (yellow Greiner) and rinse with 30 ml CPW 20S.    -   4. Transfer to 4 round-bottom tubes.    -   5. Centrifuge for 10 minutes at 600 rpm without brakes.    -   6. Collect the floating protoplasts with a pasteur-pipet and        transfer them to glass tubes containing W5.    -   7. Centrifuge for 6 min at 600 rpm with brakes.    -   8. Discard supernatant, collect the pellets in a glass tube, and        fill the tube up with 10 ml of W5. Centrifuge for 6 minutes at        600 rpm with brakes. Repeat once.    -   9. Count the protoplasts (pps) and adjust to 1,000,000 pps/ml        with MaMg.

The isolated protoplasts were then transfected using the CRISPRplasmids. The protoplast transfection protocol was as follows:

-   -   1. Add plasmid DNA, 5 μg CAS-9-2+20 g gRNA15 sgRNA to the bottom        of al0 ml red-capped round-bottom tube.    -   2. Add 0.5 ml of the 1,000,000 pps/ml MaMg preparation (see step        9 of protoplast isolation protocol), and mix gently.    -   3. Add 0.5 ml PEG4000 Fluka solution (using a P1000 with a        cut-off tip), mix immediately, and leave at room temperature for        20 min.    -   4. Add 9 ml of W5 and mix carefully. Centrifuge for 6 min at 600        rpm with brakes and repeat once.    -   5. Take up 80,000 pps/ml B-medium. Add 3 ml per 3 cm TC petri        dish and close with parafilm.    -   6. Place dishes in the dark at 25° C.

Next, the transfected protoplasts were regenerated. The cell divisionand regeneration protocol was as follows:

-   -   1. 9 days after transfection, add 1.5 ml C-medium.    -   2. 23 days after transfection, transfer calli and medium        contained in 1 small dish to 1 deep 9 cm petri dish with 15 ml        K3-soft medium.    -   3. When the calli are 3 mm in size, transfer them to K3-solid        medium for regenerating shoots.    -   4. When shoots formed transfer them to MS20 for rooting.

The composition of the media and stocks used in the protoplasttransfection and regeneration processes are shown in Tables 7A and 7B,below.

TABLE 7A Composition of media and stocks used for protoplast protocols 1l 0.6% enzyme solution^(1,3) 10x K3 stock 100 ml Cellulase onozuka R-106 g Macerozyme R-10 10 g Sucrose 136.9 g MES 586 mg 2,4-D 1 mg BAP 0.5mg CPW 20S¹ 10x CPW stock 100 ml Sucrose 200 g pH 5.8 MaMg¹ 500 mlMannitol 45 g MgCl₂•6H₂O 1520 mg MES 500 mg pH 5.6-5.8 1 l K3-soft² 10xK3 stock 100 ml Sucrose 68.4 g Sea-plaque agarose 2 g BAP 0.5 mg 2,4-D0.1 mg NAA 0.1 mg pH 5.8 K3-solid² 10x K3 stock 100 ml Sucrose 5 gSea-plaque agarose 7 g Zeatin 1 mg BAP 0.5 mg IAA 0.1 mg pH 5.8 ¹Filtersterilize; ²Autoclave; ³Store in freezer

TABLE 7B Composition of media and stocks used for protoplast protocolsTVL² g/l Sorbitol 54.65 CaCl₂•2H₂O 7.35 MES 0.586 pH 5.8 mg/l 10x K3stock^(1,3) NH₄NO₃ 2500 CaCl₂•2H₂0 3000 KNO₃ 25000 MgSO₄•7H₂0 2500(NH₄)₂SO₄ 1340 NaH₂PO₄ 1500 NaFeEDTA 430 MnSO₄•1H₂O 100 H₃BO₃ 30ZnSO₄•7H₂O 20 KI 10 Na₂MoO₄•2H₂O 2.5 CoCl₂•6H₂0 0.25 CuSO₄•5H₂O 0.25Xylose 2500 Myo inositol 1000 Thiamine•HCl 100 Nicotinic acid 10Pyridoxin•HCl 10 10x CPW stock solution^(1,3) KH₂PO₄ 272 KNO₃ 1010CaCl₂•2H₂O 14800 MgSO₄•7H₂O 2460 KI 1.6 CuSO₄•5H₂O 0.25 W5¹ g/lCaCl₂•2H₂O 18.4 NaCl 9 KCl 0.8 Glucose 1 pH 5.8 mg/l B-medium¹CaCl₂•2H₂O 750 KNO₃ 2500 MgSO₄•7H₂O 250 (NH₄)₂SO₄ 134 NaH₂PO₄ 150Na₂EDTA 37.3 FeSO₄•7H₂O 27.8 MnSO₄•1H₂O 10 H₃BO₃ 3 ZnSO₄•7H₂O 2 KI 0.75Na₂MoO₄•2H₂O 0.25 CoCl₂•6H₂O 0.025 CuSO₄•5H₂O 0.025 Myo-inositol 100Thiamine•HCl 10 Nicotinic acid 1 Pyridoxine•HCl 1 Glucose 20000 Mannitol70000 NAA 1 BAP 1 2,4-D 0.25 pH 5.8 C-medium¹ NH₄NO₃ 200 KH₂PO₄ 75CaCl₂•2H₂O 525 KNO₃ 1250 MgSO₄•7H₂O 250 (NH₄)₂SO₄ 67 NaH₂PO₄ 75 Na₂EDTA37.3 FeSO₄•7H₂0 27.8 MnSO₄•1H₂O 10 H₃BO₃ 3 ZnSO₄•7H₂O 2 KI 0.75Na₂MoO₄•2H₂O 0.25 COCl₂•6H₂O 0.025 CuSO₄•5H₂O 0.025 Myo-inositol 100Thiamine•HCl 10 Nicotinic acid 1 Pyridoxine•HCl 1 Glucose 40000 Mannitol20000 NAA 0.2 BAP 1 pH 5.8 ¹Filter sterilize; ²Autoclave; ³Do not adjustpH and store at 0-5° C.

Genotyping of Regenerated Plants

After multiple prescreening rounds of both calli and microcalli,plantlets were genotyped. DNA was isolated from the leaves of plantlets,and two independent DNA isolations were sequenced using amplicon deepsequencing (at least 100× coverage) and results were analyzed usingCRISPRESSO (http://crispresso.rocks/) with default settings. Thesequencing primers used are shown in Table 8, below, whereby primers B09and F02 were used together and primers BoDMR6.12ex3f1 BC andBoDMR6.12ex3f1 BC were used together. In addition to genotyping, ploidvof the selected plants was confirmed to be 2N via flow cytometry.

TABLE 8 Sequencing primers for plantlet genotyping Primer namePrimer sequence B09 ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCTTTTGCAGGGAAGTTGTG (SEQ ID NO: 133) F02CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTACCGCAA ACGGTAGCATCT (SEQ ID NO: 134)BoDMR6.12ex3f1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTCAACA BCCATGGCAGTCAAC (SEQ ID NO: 135) BoDMR6.12ex3r1CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTGTCGGTA BCTGAGCAGGTAAAC (SEQ ID NO: 136)

A double knockout (i.e., knockout mutations in both DMR6-1 and DMR6-2)line was generated using the above methods. FIGS. 22A-22F show thenucleotide alignment of the DMR6-1 knockout allele with thecorresponding wild type sequence of DMR6-1, whereby the knockout allele(BoDMR6_−5 bp_scf1=SEQ ID NO: 137) is shown below the wild type sequence(BoDMR6_WT_scf108=SEQ ID NO: 114). FIGS. 23A-23E show the nucleotidealignment of the DMR6-2 knockout allele with the corresponding wild typesequence of DMR6-2, whereby the knockout allele (BoDMR6_-lbp_scf1=SEQ IDNO: 138) is shown below the wild type sequence (BoDMR6_WT_scf155=SEQ IDNO: 115). FIGS. 24A-24B show the polypeptide alignment of the DMR6-1knockout polypeptide with the corresponding wild type polypeptide ofDMR6-1, whereby the knockout polypeptide (BoDMR6_−5 bp_scf1=SEQ ID NO:139) is shown below the wild type polypeptide (BoDMR6_WT_scf108=SEQ IDNO: 112). FIGS. 25A-25B show the polypeptide alignment of the DMR6-2knockout polypeptide with the corresponding wild type polypeptide ofDMR6-2, whereby the knockout polypeptide (BoDMR6_-lbp_scf1=SEQ ID NO:140) is shown below the wild type polypeptide (BoDMR6_WT_scf155=SEQ IDNO: 113). The double knockout line was referred to as “GE” for “GenomeEditing”. In addition to the GE line, a wild type line was produced fromthe same process, i.e., plants were regenerated from protoplasts thatdid not contain the double mutation. This line was referred to as“GE.WT” or “WT”.

Preparation for Xcc Test

The plants used in the Xcc test were control plants, GE plants, andGE.WT plants. The control plants were a panel of three lines: KT38(susceptible to Xcc), Resistant line 1 (R1; resistant to Xcc 1 and Xcc4), and Resistant line 2 (R2; resistant to Xcc 1 and Xcc 4). R1 and R2have been used in Brassica oleracea breeding as sources of resistance,and it is thought that their resistances are classical resistance genesthat are race-specific. The control plants were first sown, and thentransferred to 1 liter pots. These pots were placed in a cleangreenhouse for development. Plants were considered sufficiently maturefor testing when they had at least 5 to 6 true leaves.

The GE plants and GE.WT plants were produced as described above. GEplants (cuttings) from in vitro tissue culture were transferred to a GMOgreenhouse. The plants were potted in 1 liter pots, and grown in the GMOgreenhouse. Plants were considered sufficiently mature for testing whenthey had at least 5 to 6 expanded leaves.

The pathogens used in the Xcc test were Xcc race 1 (Xcc 1) and Xcc race4 (Xcc 4) isolates from the pathogen collection. If there were doubtsabout the purity of the isolate, it was plated on semi-selective mediato check whether it was clean before beginning the preparation. Forpreparation, the chosen isolate was plated on KB plates and grown for 2days. Then, the bacteria were washed off of the plates and diluted withnormal tap water to an OD of 0.4. In these tests, Xcc 1 and Xcc 4 wereused, as they are the most prevalent in B. oleracea growing areas, butXcc 1 and Xcc 6 would have been another option (Vicente et al. (2013),Molecular Plant Pathology, 14(1): 2-18).

Xcc Test Protocol

For testing, a climate cell or greenhouse was used that was kept at 25°C. with a day-night cycle of 16 hours light-8 hours dark (16:8 cycle).In this climate cell or greenhouse, a tent was built to cover the plantsso that they could be maintained at high humidity. Inside the tent,containers were used to create a surface for the plants to stand in thatcould be filled with water. The day before infection, plants were placedin the containers, and the containers were filled with water so that theplants were standing in a shallow layer of water. At this point, a ringwas placed on the youngest formed true leaf of the plants if needed.

The plants were infected by spraying them with a fine mist of thebacterial suspension (preparation described above). Then, the tent wasclosed to maintain humidity. The day after infection, the tent wasslightly opened to avoid secondary infections. In order to keep thehumidity high during the test, a layer of water was kept in thecontainers during the first 2 to 3 weeks. After that, the water layerwas not maintained so that secondary infections and/or rotting would notoccur.

Scoring of local infection began as soon as the first symptoms appeared.Local infection symptoms were scored for all of the leaves that werefully developed at the time of infection. Scoring of systemic infectionwas done one, two, and three weeks after infection. Systemic infectionsymptoms were scored for any new leaves that emerged after theinfection.

For both local and systemic infections, a scale of 1 to 9 was used toscore the severity of infection. Table 9 provides a detailed descriptionof the Xcc test scoring scale. A score of 9 corresponded to a plant thatwas completely healthy with an absence of symptoms. A score of 1corresponded to a plant that was completely wilted/dead. At the end ofsystemic infection scoring, susceptible plants scored 1. FIGS. 18A-18Dshow exemplary images of plants scored as 1, 3, 5, and 7.

TABLE 9 Xcc test scoring scale Local infection Systemic infectionInfection Infection severity Phenotype severity Phenotype 1 All leavesare more than 1 Plant is dead 50% infected 3 All leaves are less than50% 3 All new leaves are infected infected 4 More than 2, but not all 4More than 2, but not all new leaves infected leaves infected 5 1 or 2leaves infected with 5 1 or 2 new leaves infected with V-shaped lesionsV-shaped lesions 7 No clear infection, but some 7 No clear infection,but older leaves show spots leaves show some symptoms 9 Nothing to see,completely 9 Nothing to see, completely clean plant clean plant

Downy Mildew Whole Plant Test G

The downy mildew whole plant test was performed as described in Example7.

Results

FIGS. 19A and 19B show the results of the Xcc test using Xcc 1. FIG. 19Ashows local infection scores. Both of the resistant control plant lines(R1 and R2) had an average local infection score of about 7, while thesusceptible control plant line (KT38) had an average local infectionscore of about 2. The wild type plants processed with the same protocolas the GE plants (GE.WT) had an average local infection score of about3. The DMR6-1 and DMR6-2 double knockout plants (GE) had an averagelocal infection score of about 5, which was intermediate between theresistant and susceptible control line results. FIG. 19B shows systemicinfection scores. Both R1 and R2 had an average systemic infection scoreof about 5 to 6, while KT38 had an average systemic infection score ofabout 1 to 2. GE.WT had an average systemic infection score of about 3.GE had an average systemic infection score of about 6, which wascomparable to the resistant control line (R1 and R2) results.

FIGS. 19C and 19D show the results of the Xcc test using Xcc 4. FIG. 19Cshows local infection scores. The resistant control line R1 had anaverage local infection score of about 5, while the resistant controlline R2 had an average local infection score of about 8. The susceptiblecontrol line (KT38) had an average local infection score of about 1.GE.WT had an average local infection score of about 3. GE had an averagelocal infection score of about 5, which was the same as the R1 score,and intermediate between the R2 and KT38 scores. FIG. 19D shows systemicinfection scores. Both R1 and R2 had an average systemic infection scoreof about 8, while KT38 had an average systemic infection score ofabout 1. GE.WT had an average systemic infection score of about 4, andGE had an average systemic infection score of about 7. FIG. 20 shows anexemplary image of the systemic resistance phenotype of a GE plant (onright) compared to a GE.WT plant (on left).

For both Xcc 1 and Xcc 4, the local infection score results showed thatGE plants were more resistant than GE.WT and KT38 plants. The resistantcontrol plants, R1 and R2, were more resistant than GE plants. Thesystemic infection score results for both Xcc 1 and Xcc 4 showed that GEplants were more resistant than GE.WT and KT38. The comparison to theresistant controls revealed differences between Xcc 1 and Xcc 4: R1 andR2 plants were more resistant to Xcc 4 than GE plants, but GE, R1, andR2 plants all had a similar resistance to Xcc 1.

FIG. 21 shows the result of the downy mildew whole plant test. WT plantshad an average disease score of about 5 (i.e., susceptible), while GEplants had an average disease score of about 7 (i.e., resistant).

Taken together, these results show that DMR6-1 and DMR6-2 doubleknockout plants have intermediate resistance to Xcc (i.e., Xcc 1 and Xcc4) and resistance to downy mildew.

Example 9 Testing a Double Knockout of DMR6-1 and DMR6-2 in BrassicaSpecies for Resistance to Downy Mildew and Xcc Experimental Procedures

Additional cauliflower lines, broccoli lines, and kohlrabi linescontaining a double knockout of DMR6-1 and DMR6-2 will be produced usingthe mutant alleles described in Examples 7 and 8. These double knockoutlines will be tested for resistance to downy mildew and Xcc as describedin Examples 7 and 8.

Successful lines will be combined with resistant QTL identified fromknown resistant lines R1 and R2. These lines containing stackedresistant traits will be developed commercially.

Results

The cauliflower lines, broccoli lines, and kohlrabi lines containing adouble knockout of DMR6-1 and DMR6-2 are resistant to downy mildew andare intermediate resistant to Xcc. The lines containing stackedresistant traits are resistant to downy mildew and are intermediateresistant to Xcc.

What is claimed is:
 1. A mutant Brassica plant, wherein the plant has areduced activity or a reduced level of a DMR6-1 polypeptide as comparedto a corresponding wild type Brassica plant, and wherein the mutantBrassica plant has at least one non-natural mutation introduced into theDMR6-1 gene; and wherein the mutant Brassica plant has a reducedactivity or a reduced level of a DMR6-2 polypeptide as compared to acorresponding wild type Brassica plant, and wherein the mutant Brassicaplant has at least one non-natural mutation introduced into the DMR6-2gene.
 2. The mutant Brassica plant of claim 1, wherein the plantexhibits resistance selected from the group consisting of resistance toHyaloperonospora parasitica/Hyaloperonospora brassicae, intermediateresistance to Xanthomonas campestris campestris, and any combinationthereof.
 3. The mutant Brassica plant of claim 2, wherein the DMR6-1polypeptide is selected from the group consisting of a polypeptide withat least 85% sequence identity, at least 88% sequence identity, at least90% sequence identity, at least 95% sequence identity, and at least 99%sequence identity to SEQ ID NO: 112; and wherein the DMR6-2 polypeptideis a polypeptide selected from the group consisting of a protein with atleast 85% sequence identity, at least 88% sequence identity, at least90% sequence identity, at least 95% sequence identity, and at least 99%sequence identity to SEQ ID NO:
 113. 4. The mutant Brassica plant ofclaim 3, wherein the DMR6-1 gene is selected from the group consistingof a nucleotide with at least 85% sequence identity, at least 88%sequence identity, at least 90% sequence identity, at least 95% sequenceidentity, and at least 99% sequence identity to SEQ ID NO: 114; andwherein the DMR6-2 gene is selected from the group consisting of anucleotide with at least 85% sequence identity, at least 88% sequenceidentity, at least 90% sequence identity, at least 95% sequenceidentity, and at least 99% sequence identity to SEQ ID NO:
 115. 5. Themutant Brassica plant of claim 4, wherein the non-natural mutation inDMR6-1 is a premature stop codon, and wherein the non-natural mutationin DMR6-2 is a premature stop codon.
 6. The mutant Brassica plant ofclaim 2, wherein the at least one non-natural mutation introduced intothe DMR6-1 gene reduces an activity or a level of a DMR6-1 polypeptideas compared to a corresponding wild type Brassica plant; and wherein theat least one non-natural mutation introduced into the DMR6-2 genereduces an activity or a level of a DMR6-2 polypeptide as compared to acorresponding wild type Brassica plant.
 7. The mutant Brassica plant ofclaim 6, wherein the DMR6-1 polypeptide is selected from the groupconsisting of a polypeptide with at least 85% sequence identity, atleast 88% sequence identity, at least 90% sequence identity, at least95% sequence identity, and at least 99% sequence identity to SEQ ID NO:112; and wherein the DMR6-2 polypeptide is a polypeptide selected fromthe group consisting of a protein with at least 85% sequence identity,at least 88% sequence identity, at least 90% sequence identity, at least95% sequence identity, and at least 99% sequence identity to SEQ ID NO:113.
 8. The mutant Brassica plant of claim 7, wherein the DMR6-1 gene isselected from the group consisting of a nucleotide with at least 85%sequence identity, at least 88% sequence identity, at least 90% sequenceidentity, at least 95% sequence identity, and at least 99% sequenceidentity to SEQ ID NO: 114; and wherein the DMR6-2 gene is selected fromthe group consisting of a nucleotide with at least 85% sequenceidentity, at least 88% sequence identity, at least 90% sequenceidentity, at least 95% sequence identity, and at least 99% sequenceidentity to SEQ ID NO:
 115. 9. The mutant Brassica plant of claim 8,wherein the at least one non-natural mutation that reduces the activityor level of the DMR6-1 protein is selected from the group consisting ofa premature stop codon introduced into the DMR6-1 coding sequence, aframeshift mutation introduced into the DMR6-1 coding sequence, aninsertion introduced into the DMR6-1 coding sequence, a deletion of apart or a whole of the DMR6-1 coding sequence, an altered amino acid ina conserved domain of the DMR6-1 protein, a modified upstream sequence,a mutated promoter element, and an activated repressor element; andwherein the at least one non-natural mutation that reduces the activityor level of the DMR6-2 protein is selected from the group consisting ofa premature stop codon introduced into the DMR6-2 coding sequence, aframeshift mutation introduced into the DMR6-2 coding sequence, aninsertion introduced into the DMR6-2 coding sequence, a deletion of apart or a whole of the DMR6-2 coding sequence, an altered amino acid ina conserved domain of the DMR6-2 protein, a modified upstream sequence,a mutated promoter element, and an activated repressor element.
 10. Themutant Brassica plant of claim 9, wherein the plant is a Brassicaoleracea cultivar selected from the group consisting of cabbage,kohlrabi, kale, Brussels sprouts, collard greens, broccoli, Romanescobroccoli, and cauliflower.
 11. The mutant Brassica plant of claim 2,wherein the non-natural mutation introduced into the DMR6-1 gene isselected from the group consisting of a C to T mutation at a positioncorresponding to nucleotide 181 of reference sequence SEQ ID NO: 114, aC to T mutation at a position corresponding to nucleotide 691 ofreference sequence SEQ ID NO: 114, a G to A mutation at a positioncorresponding to nucleotide 714 of reference sequence SEQ ID NO: 114,and a deletion of five nucleotides starting at a position correspondingto nucleotide 601 of reference sequence SEQ ID NO: 114; and wherein thenon-natural mutation introduced into the DMR6-2 gene is selected fromthe group consisting of a G to A mutation at a position corresponding tonucleotide 368 of reference sequence SEQ ID NO: 115, and a deletion ofthe nucleotide at a position corresponding to nucleotide 600 ofreference sequence SEQ ID NO:
 115. 12. A seed, tissue, or plant part ofthe Brassica plant of claim 2, wherein the seed, tissue, or plant partcomprises a reduced activity or a reduced level of a DMR6-1 polypeptideas compared to a corresponding wild type Brassica plant and a reducedactivity or a reduced level of a DMR6-2 polypeptide as compared to acorresponding wild type Brassica plant, and wherein the seed, tissue, orplant part comprises at least one non-natural mutation in the DMR6-1gene and at least one non-natural mutation in the DMR6-2 gene.
 13. Amethod for obtaining a mutant Brassica plant comprising: introducing atleast one non-natural mutation into the DMR6-1 nucleotide codingsequence and introducing at least one non-natural mutation into theDMR6-2 nucleotide coding sequence to produce a mutant Brassica plantwith a reduced activity or a reduced level of a DMR6-1 polypeptide ascompared to a corresponding wild type Brassica plant and a reducedactivity or a reduced level of a DMR6-2 polypeptide as compared to acorresponding wild type Brassica plant.
 14. The method of claim 13,wherein the mutant Brassica plant exhibits resistance selected from thegroup consisting of resistance to Hyaloperonosporaparasitica/Hyaloperonospora brassicae, intermediate resistance toXanthomonas campestris campestris, and any combination thereof.
 15. Themethod of claim 14, wherein the non-natural mutation is achieved by amutagenic treatment, a radiation treatment, or a gene editing technique.16. A mutant Brassica plant produced from the method of claim 14,wherein the plant comprises at least one non-natural mutation in theDMR6-1 gene and at least one non-natural mutation in the DMR6-2 gene,wherein the plant further comprises a reduced activity or a reducedlevel of a DMR6-1 polypeptide as compared to a corresponding wild typeBrassica plant and a reduced activity or a reduced level of a DMR6-2polypeptide as compared to a corresponding wild type Brassica plant, andwherein the plant exhibits resistance selected from the group consistingof resistance to Hyaloperonospora parasitica/Hyaloperonospora brassicae,intermediate resistance to Xanthomonas campestris campestris, and anycombination thereof.
 17. A mutant Brassica plant produced from themethod of claim 16, wherein the plant comprises at least one non-naturalmutation in the DMR6-1 nucleotide coding sequence that reduces anactivity or a level of a DMR6-1 polypeptide as compared to acorresponding wild type Brassica plant; wherein the plant comprises atleast one non-natural mutation in the DMR6-2 nucleotide coding sequencethat reduces an activity or a level of a DMR6-2 polypeptide as comparedto a corresponding wild type Brassica plant; and wherein the plantexhibits resistance selected from the group consisting of resistance toHyaloperonospora parasitica/Hyaloperonospora brassicae, intermediateresistance to Xanthomonas campestris campestris, and any combinationthereof.
 18. The mutant Brassica plant of claim 17, wherein the DMR6-1polypeptide is selected from the group consisting of a polypeptide withat least 85% sequence identity, at least 88% sequence identity, at least90% sequence identity, at least 95% sequence identity, at least 99%sequence identity to SEQ ID NO: 112; and wherein the DMR6-2 polypeptideis a polypeptide selected from the group consisting of a protein with atleast 85% sequence identity, at least 88% sequence identity, at least90% sequence identity, at least 95% sequence identity, and at least 99%sequence identity to SEQ ID NO:
 113. 19. The mutant Brassica plant ofclaim 18, wherein the DMR6-1 gene is selected from the group consistingof a nucleotide with at least 85% sequence identity, at least 88%sequence identity, at least 90% sequence identity, at least 95% sequenceidentity, and at least 99% sequence identity to SEQ ID NO: 114; andwherein the DMR6-2 gene is selected from the group consisting of anucleotide with at least 85% sequence identity, at least 88% sequenceidentity, at least 90% sequence identity, at least 95% sequenceidentity, and at least 99% sequence identity to SEQ ID NO:
 115. 20. Aseed, tissue, or plant part of the mutant Brassica plant of claim 19.